Compositions and methods for enhancing drug delivery across and into epithelial tissues

ABSTRACT

This invention provides compositions and methods for enhancing delivery of drugs and other agents across epithelial tissues, including the skin, gastrointestinal tract, pulmonary epithelium, ocular tissues and the like. The compositions and methods are also useful for delivery across endothelial tissues, including the blood brain barrier. The compositions and methods employ a delivery enhancing transporter that has sufficient guanidino or amidino sidechain moieties to enhance delivery of a compound conjugated to the reagent across one or more layers of the tissue, compared to the non-conjugated compound. The delivery-enhancing polymers include, for example, poly-arginine molecules that are preferably between about 6 and 25 residues in length.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/542,278, filed Oct. 2, 2006, which is a continuation of U.S. patentapplication Ser. No. 10/740,365, filed Dec. 17, 2003, which is acontinuation of U.S. patent application Ser. No. 09/792,480, filed Feb.23, 2001, now U.S. Pat. No. 6,669,951, which is a continuation-in-partapplication of U.S. patent application Ser. No. 09/648,400, filed Aug.24, 2000, now U.S. Pat. No. 6,593,292, which claims priority to U.S.Provisional Patent Application No. 60/150,510, filed Aug. 24, 1999. Thedisclosures of each of these applications are incorporated herein byreference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to the field of compositions and methods thatenhance the delivery of drugs and other compounds across the dermal andepithelial membranes, including, for example, skin, the gastrointestinalepithelium and the bronchial epithelium.

2. Background

Transdermal or transmucosal drug delivery is an attractive route of drugdelivery for several reasons. Gastrointestinal drug degradation and thehepatic first-pass effect are avoided. In addition, transdermal andtransmucosal drug delivery is well-suited to controlled, sustaineddelivery (see, e.g., Elias, In Percutaneous Absorption:Mechanisms-Methodology-Drug Delivery, Bronaugh & Maibach, Eds., pp 1-12,Marcel Dekker, New York, 1989.). For many applications, traditionalmethods of administering drugs are not optimal because of the very largeinitial concentration of the drug. Transdermal delivery could allow amore uniform, slower rate of delivery of a drug. Moreover, patientcompliance is encouraged because such delivery methods are easy to use,comfortable, convenient and non-invasive.

These advantages of transdermal and transmucosal delivery have not ledto many clinical applications because of the low permeability ofepithelial membranes, the skin in particular, to drugs. The difficultiesin delivering drugs across the skin result from the barrier property ofskin. Skin is a structurally complex thick membrane that represents thebody's border to the external hostile environment. The skin is composedof the epidermis, the dermis, the hypodermis, and the adenexalstructures (epidermal appendages). The epidermis, the outermostepithelial tissue of the skin, is itself composed of several layers—thestratum corneum, the stratum granulosum, the stratum spinosum, and thestratum basale.

Compounds that move from the environment into and through intact skinmust first penetrate the stratum corneum, the outermost layer of skin,which is compact and highly keratinized. The stratum corneum is composedof several layers of keratin-filled skin cells that are tightly boundtogether by a “glue” composed of cholesterol and fatty acids. Thethickness of the stratum corneum varies depending upon body location. Itis the presence of stratum corneum that results in the impermeability ofthe skin to pharmaceutical agents. The stratum corneum is formednaturally by cells migrating from the basal layer toward the skinsurface where they are eventually sloughed off. As the cells progresstoward the surface, they become progressively more dehydrated andkeratinized. The penetration across the stratum corneum layer isgenerally the rate-limiting step of drug permeation across skin. See,e.g., Flynn, G. L., In Percutaneous Absorption:Mechanisms-Methodology-Drug Delivery, supra, at pages 27-53.

After penetration through the stratum corneum layer, systemically actingdrug molecules then must pass into and through the epidermis; thedermis, and finally through the capillary walls of the bloodstream. Theepidermis, which lies under the stratum corneum, is composed of threelayers. The outermost of these layers is the stratum granulosum, whichlies adjacent to the stratum corneum, is composed of cells that aredifferentiated from basal cells and keratinocytes, which make up theunderlying layers. having acquired additional keratin and a moreflattened shape. The cells of this layer of the epidermis, which containgranules that are composed largely of the protein filaggrin. Thisprotein is believed to bind to the keratin filaments to form the keratincomplex. The cells also synthesize lipids that function as a “cement” tohold the cells together. The epidermis, in particular the stratumgranulosum, contains enzymes such as aminopeptidases.

The next-outermost layer of the epidermis is the stratum spinosum, theprincipal cells of which are keratinocytes, which are derived from basalcells that comprise the basal cell layer. Langerhans cells, which arealso found in the stratum spinosum, are antigen-presenting cells andthus are involved in the mounting of an immune response against antigensthat pass into the skin. The cells of this layer are generally involvedIn contact sensitivity dermatitis.

The innermost epidermal layer is the stratum basale, or basal celllayer, which consists of one cell layer of cuboidal cells that areattached by hemi-desmosomes to a thin basement membrane which separatesthe basal cell layer from the underlying dermis. The cells of the basallayer are relatively undifferentiated, proliferating cells that serve asa progenitor of the outer layers of the epidermis. The basal cell layerincludes, in addition to the basal cells, melanocytes.

The dermis is found under the epidermis, which is separated from thedermis by a basement membrane that consists of interlocking rete ridgesand dermal papillae. The dermis itself is composed of two layers, thepapillary dermis and the reticular dermis. The dermis consists offibroblasts, histiocytes, endothelial cells, perivascular macrophagesand dendritic cells, mast cells, smooth muscle cells, and cells ofperipheral nerves and their end-organ receptors. The dermis alsoincludes fibrous materials such as collagen and reticulin, as well as aground substance (principally glycosaminoglycans, including hyaluronicacid, chondroitin sulfate, and dermatan sulfate).

Several methods have been proposed to enhance transdermal transport ofdrugs. For example, chemical enhancers (Burnette, R. R. In DevelopmentalIssues and Research Initiatives; Hadgraft J., Ed., Marcel Dekker: 1989;pp. 247-288), iontophoresis, and others have been used. However, inspite of the more than thirty years of research that has gone intodelivery of drugs across the skin in particular, fewer than a dozendrugs are now available for transdermal administration in, for example,skin patches.

Transport of drugs and other molecules across the blood-brain bather isalso problematic. The brain capillaries that make up the blood-brainbather are composed of endothelial cells that form tight junctionsbetween themselves (Goldstein et al., Scientific American 255:74-83(1986); Pardridge, W. M., Endocrin. Rev. 7: 314-330 (1986)). Theendothelial cells and the tight intercellular junctions that join thecells form a barrier against the passive movement of many molecules fromthe blood to the brain. The endothelial cells of the blood-brain barrierhave few pinocytotic vesicles, which in other tissues can allow somewhatunselective transport across the capillary wall. Nor is the blood-brainbarrier interrupted by continuous gaps or channels that run through thecells, thus allowing for unrestrained passage of drugs and othermolecules.

Thus, a need exists for improved reagents and methods for enhancingdelivery of compounds, including drugs, across epithelial tissues andendothelial tissues such as the skin, the gastrointestinal tract, theeye and the blood-brain barrier. The present invention fulfills this andother needs.

SUMMARY OF THE INVENTION

The present invention provides methods of targeting a compound to agastrointestinal epithelium of an animal. The methods involveadministering to the gastrointestinal epithelium a conjugate thatincludes the compound and a delivery-enhancing transporter. Thedelivery-enhancing transporters, which are also provided by theinvention, have sufficient guanidino or amidino moieties to increasedelivery of the conjugate into the gastrointestinal epithelium or oculartissues compared to delivery of the compound in the absence of thedelivery-enhancing transporter. In some embodiments, delivery of theconjugate into the gastrointestinal epithelium or ocular tissue isincreased at least two-fold compared to delivery of the compound in theabsence of the delivery-enhancing transporter. In more preferredembodiments, delivery of the conjugate into the gastrointestinalepithelium is increased at least ten-fold compared to delivery of thecompound in the absence of the delivery-enhancing transporter. Thedelivery-enhancing transporter and the compound are typically attachedthrough a linker. In addition, the conjugate can comprise two or moredelivery-enhancing transporters linked to the compound.

Typically, the delivery-enhancing transporters comprise fewer than 50subunits and comprise at least 6 guanidino or amidino moieties. In someembodiments, the subunits are amino acids. In some embodiments, thedelivery-enhancing transporters have from 6 to 25 guanidino or amidinomoieties, and more preferably between 7 and 15 guanidino moieties andstill more preferably, at least six contiguous guanidino and/or amidinomoieties. In some embodiments, the delivery-enhancing transportersconsist essentially of 5 to 50 subunits, at least 50 of which compriseguanidino or amidino residues. In some of these embodiments, thesubunits are natural or non-natural amino acids. For example, in someembodiments, the delivery-enhancing transporter comprises 5 to 25arginine residues or analogs thereof. For example, the transporter cancomprise seven contiguous D-arginines.

In some embodiments, the delivery-enhancing transporter comprises 7-15arginine residues or analogs of arginine. The delivery-enhancingtransporter can have at least one arginine that is a D-arginine and insome embodiments, all arginines are D-arginine. In some embodiments, atleast 70% of the amino acids are arginines or arginine analogs. In someembodiments, the delivery-enhancing transporter comprises at least 5contiguous arginines or arginine analogs. The delivery-enhancingtransporters can comprise non-peptide backbones. In addition, in someaspects, the transporter is not attached to an amino acid sequence towhich the delivery-enhancing molecule is attached in a naturallyoccurring protein.

The delivery-enhancing transporters and methods of the invention areuseful for delivering drugs, diagnostic agents, and other compounds ofinterest to the gastrointestinal epithelium. The methods andcompositions of the invention can be used not only to deliver thecompounds to the particular site of administration, but also providesystemic delivery. In some embodiments, the conjugate is administeredbucally or as a suppository. The compounds of the conjugate can be atherapeutic for a disease such as inflammatory bowel disease, coloncancer, ulcerative colitis, gastrointestinal ulcers, constipation andimbalance of salt and water absorption. Thus, the compounds can includeimmunosuppressives, ascomycins, corticosteroids, laxatives, antibioticsor anti-neoplastic agents. In some aspects of the invention, thecompound is targeted to the iliem and/or colon.

The delivery-enhancing transporters and methods of the invention areuseful for delivering drugs, diagnostic agents, and other compounds ofinterest to the eye and other ocular tissues. In some embodiments, theconjugate is administered as eye drops or as an injection. The compoundsof the conjugate include therapeutics for a disease such asconjunctivitis, bacterial infections, viral infections, dry eye andglaucoma. Thus, the compounds can include antibacterial compounds,antiviral compounds, cyclosporin, ascomycins and corticosteroids.

As discussed above, the compound to be delivered can be connected to thedelivery-enhancing transporter by a linker. In some embodiments, thelinker is a releasable linker which releases the compound, inbiologically active form, from the delivery-enhancing transporter afterthe compound has passed into and/or through one or more layers of theepithelial and/or endothelial tissue. In some embodiments, the compoundis released from the linker by solvent-mediated cleavage. The conjugateis, in some embodiments, substantially stable at acidic pH but thecompound is substantially released from the delivery-enhancingtransporter at physiological pH. In some embodiments, the half-life ofthe conjugate is between 5 minutes and 24 hours upon contact with theskin or other epithelial or endothelial tissue. For example, thehalf-life can be between 30 minutes and 2 hours upon contact with theskin or other epithelial or endothelial tissue.

Examples of conjugate structures of the invention include those havingstructures such as 3, 4, or 5, as follows:

wherein R¹ comprises the compound; X is a linkage formed between afunctional group on the biologically active compound and a terminalfunctional group on the linking moiety; Y is a linkage formed from afunctional group on the transport moiety and a functional group on thelinking moiety; A is N or CH; R² is hydrogen, alkyl, aryl, acyl, orallyl; R³ comprises the delivery-enhancing transporter; R⁴ is S, O, NR⁶or CR⁷R⁸; R⁵ is H, OH, SH or NBR⁶; R⁶ is hydrogen, alkyl, aryl, acyl orallyl; k and m are each independently selected from 1 and 2; and n is 1to 10.

Preferably, X is selected from the group consisting of —C(O)O—,—C(O)NH—, —OC(O)NH—, —S—S—, —C(S)O—, —C(S)NH—, —NHC(O)NH—, —SO₂NH—,—SONH—, phosphate, phosphonate phosphinate, and CR⁷R⁸, wherein R⁷ and R⁸are each independently selected from the group consisting of H andalkyl. In some embodiments, R₄ is S; R₅ is NHR₆; and R₆ is hydrogen,methyl, allyl, butyl or phenyl. In some embodiments, R₂ is benzyl; k, m,and n are each 1, and X is O. In some embodiments, the conjugate,comprises structure 3, Y is N, and R² is methyl, ethyl, propyl, butyl,allyl, benzyl or phenyl. In some embodiments, R² is benzyl; k, m, and nare each 1, and X is —OC(O)—. In some embodiments, the conjugatecomprises structure 4; R⁴ is S; R⁵ is NHR⁶; and R⁶ is hydrogen, methyl,allyl, butyl or phenyl. In some embodiments, the conjugate comprisesstructure 4; R⁵ is NHR⁶; R⁶ is hydrogen, methyl, allyl, butyl or phenyl;and k and m are each 1.

The invention also provides conjugates in which the release of thelinker from the biological agent involves a first, rate-limitingintramolecular reaction, followed by a faster intramolecular reactionthat results in release of the linker. The rate-limiting reaction can,by appropriate choice of substituents of the linker, be made to bestable at a pH that is higher or lower than physiological pH. However,once the conjugate has passed into and across one or more layers of anepithelial or endothelial tissue, the linker will be cleaved from theagent. An example of a compound that has this type of linker isstructure 6, as follows:

wherein R¹ comprises the compound; X is a linkage formed between afunctional group on the biologically active compound and a terminalfunctional group on the linking moiety; Y is a linkage formed from afunctional group on the transport moiety and a functional group on thelinking moiety; Ar is an aryl group having the attached radicalsarranged in an ortho or para configuration, which aryl group can besubstituted or unsubstituted; R³ comprises the delivery-enhancingtransporter; R⁴ is S, O, NR⁶ or CR⁷R⁸; R⁵ is H, OH, SH or NHR₆; R⁶ ishydrogen, alkyl, aryl, arylalkyl, acyl or allyl; R⁷ and R⁸ areindependently selected from hydrogen or alkyl; and k and m are eachindependently selected from 1 and 2.

In some embodiments, X is selected from the group consisting of —C(O)O—,—C(O)NH—, —OC(O)NH—, —S—S—, —C(S)O—, —C(S)NH—, —SO₂NH—, —SONH—,phosphate, phosphonate phosphinate, and CR⁷R⁸, wherein R⁷ and R⁸ areeach independently selected from the group consisting of H and alkyl. Insome embodiments, R⁴ is S; R⁶ is NHR⁶; and R⁶ is hydrogen, methyl,allyl, butyl or phenyl.

In preferred embodiments, the compositions of the invention comprise alinker susceptible to solvent-mediated cleavage. For example, apreferred linker is substantially stable at acidic pH but issubstantially cleaved at physiological pH.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a reaction scheme for the preparation of an α-chloroacetylcyclosporin A derivative.

FIG. 2 shows a general procedure for the coupling of cysteine-containingpeptides to the α-chloro acetyl cyclosporin A derivative.

FIG. 3 shows a reaction scheme for the coupling of the cyclosporin Aderivative to a biotin-labeled peptide.

FIG. 4 shows a reaction scheme for coupling of a cyclosporin Aderivative to an unlabeled peptide.

FIG. 5 A-H show various types of cleavable linkers that can be used tolink a delivery-enhancing transporter to a biologically active agent orother molecule of interest. FIG. 5A shows an example of a disulfidelinkage. FIG. 5B shows a photocleavable linker which is cleaved uponexposure to electromagnetic radiation. FIG. 5C shows a modified lysylresidue used as a cleavable linker. FIG. 5D shows a conjugate in whichthe delivery-enhancing transporter T is linked to the 2′-oxygen of theanticancer agent, paclitaxel. The linking moiety includes (i) a nitrogenatom attached to the delivery-enhancing transporter, (ii) a phosphatemonoester located para to the nitrogen atom, and (iii) a carboxymethylgroup meta to the nitrogen atom, which is joined to the 2′-oxygen ofpaclitaxel by a carboxylate ester linkage. FIG. 5E a linkage of adelivery-enhancing transporter to a biologically active agent, e.g.,paclitaxel, by an aminoalkyl carboxylic acid; a linker amino group isjoined to a delivery-enhancing transporter by an amide linkage and to apaclitaxel moiety by an ester linkage. FIGS. 5F and G show chemicalstructures and conventional numbering of constituent backbone atoms forpaclitaxel and “TAXOTERE™” (R′═H, R″═BOC). FIG. 5G shows the generalchemical structure and ring atom numbering for taxoid compounds.

FIG. 6 displays a synthetic scheme for a chemical conjugate between aheptamer of L-arginine and cyclosporin A (panel A) and its pH dependentchemical release (panel B). The α-chloro ester (6i) was treated withbenzylamine in the presence of sodium iodide to effect substitution,giving the secondary amine (6ii). Amine (6ii) was treated with anhydride(6) and the resultant crude acid (6iii) was converted to itscorresponding NHS ester (6iv). Ester (6iv) was then coupled with theamino terminus of hepta-L-arginine, giving the N-Boc protected CsAconjugate (6v). Finally, removal of the Boc protecting group with formicacid afforded the conjugate (6vi) as its octatrifluoroacetate salt afterHPLC purification.

FIG. 7 displays inhibition of inflammation in murine contact dermatitisby releasable R7 CsA. Balb/c (6-7 weeks) mice were painted on theabdomen with 100 μl of 0.7% DNFB in acetone olive oil (95:5). Three dayslater both ears of the animals were restimulated with 0.5% DNFB inacetone. Mice were treated one, five, and twenty hours afterrestimulation with either vehicle alone, 1% R7 peptide alone, 1% CsA, 1%nonreleasable R7CsA, 0.01%/0.1%/1.0% releasable R7 CsA, and thefluorinated steroid positive control 0.1% triamcinolone acetonide. Earinflammation was measured 24 hours after restimulation using a springloaded caliper. The percent reduction of inflammation was calculatedusing the following formula (t−n)/(u−n), where t=thickness of thetreated ear, n=the thickness of a normal untreated ear, and u=thicknessof an inflamed ear without any treatment. N=20 animals in each group.

FIG. 8 shows a procedure for the preparation of acopper-diethylene-triaminepentaacetic acid complex (Cu-DTPA).

FIG. 9 shows a procedure for linking the Cu-DTPA to a transporterthrough an aminocaproic acid.

FIG. 10 shows a reaction for the acylation of hydrocortisone withchloroacetic anhydride.

FIG. 11 shows a reaction for linking the acylated hydrocortisone to atransporter.

FIG. 12 shows a reaction for preparation of C-2′ derivatives of taxol.

FIG. 13 shows a schematic of a reaction for coupling of a taxolderivative to a biotin-labeled peptide.

FIG. 14 shows a reaction for coupling of an unlabeled peptide to a C-2′derivative of taxol.

FIG. 15A-C shows a reaction scheme for the formation of other C-2′taxol-peptide conjugates.

FIG. 16 shows a general strategy for synthesis of a conjugate in which adrug or other biological agent is linked to a delivery-enhancingtransporter by a pH-releasable linker.

FIG. 17 shows a schematic diagram of a protocol for synthesizing a taxol2′-chloroacetyl derivative.

FIG. 18 shows a strategy by which a taxol 2′-chloroacetyl derivative islinked to an arginine heptamer delivery-enhancing transporter.

FIG. 19 shows three additional taxol-r7 conjugates that can be madeusing the reaction conditions illustrated in FIG. 18.

FIG. 20 shows the results of a 3 day MTT cytotoxicity assay using taxoland two different linkers.

FIG. 21 shows the FACS cellular uptake assay of truncated analogs ofTat₄₉₋₅₇ (Fl-ahx-RKKRRQRRR): Tat₄₉₋₅₆ (Fl-ahx-RKKRRQRR), Tat₄₉₋₅₅(Fl-ahx-RKICRRQR), Tat₅₀₋₅₇ (Fl-ahx-KKRRQRRR), and Tat₅₁₋₅₇(Fl-ahx-KRRQRRR). Jurkat cells were incubated with varyingconcentrations (12.5 μM shown) of peptides for 15 min at 23° C.

FIG. 22 shows FACS cellular uptake assay of alanine-substituted analogsof Tat₄₉₋₅₇: A-49 (Fl-ahx-AKKRRQRRR), A-50 (Fl-ahx-RAKRRQRRR), A-51(Fl-ahx-RKARRQRRR), A-52 (Fl-ahx-RKKARQRRR), A-53 (Fl-ahx-RKKRAQRRR),A-54 (Fl-ahx-RKKRRARRR), A-55 (Fl-ahx-RKKRRQARR), A-56(Fl-ahx-RKKRRQRAR), and A-57 (Fl-ahx-RKKRRQRRA). Jurkat cells wereincubated with varying concentrations (12.5 μM shown) of peptides for 12min at 23° C.

FIG. 23 shows the FACS cellular uptake assay of d- and retro-isomers ofTat₄₉₋₅₇: d-Tat49-57 (Fl-ahx-rkkrrqrrr), Tat57-49 (Fl-ahx-RRRQRRKKR),and d-Tat57-49 (Fl-ahx-rrrqrrkkr). Jurkat cells were incubated withvarying concentrations (12.5 μM shown) of peptides for 15 at 23° C.

FIG. 24 shows the FACS cellular uptake of a series of arginine oligomersand Tat₄₉₋₅₇: R5 (Fl-ahx-RRRRR), R6 (Fl-ahx-RRRRRR), R7(Fl-ahx-RRRRRRR), R8 (Fl-ahx-RRRRRRRR), R9 (Fl-ahx-RRRRRRRRR), r5(Fl-ahx-rrrr), r6 (Fl-ahx-rrrrrr), r7 (Fl-ahx-rrrrrrr), r8(Fl-ahx-rrrrrrrrrr), r9 (Fl-ahx-rrrrrrrrr). Jurkat cells were incubatedwith varying concentrations (12.5 μM shown) of peptides for 4 min at 23°C.

FIG. 25 displays the preparation of guanidine-substituted peptoids.

FIG. 26 displays the FACS cellular uptake of polyguanidine peptoids andd-arginine oligomers. Jurkat cells were incubated with varyingconcentrations (12.5 μM shown) of peptoids and peptides for 4 min at 23°C.

FIG. 27 displays the FACS cellular uptake of d-arginine oligomers andpolyguanidine peptoids. Jurkat cells were incubated with varyingconcentrations (12.5 μM shown) of fluorescently labeled peptoids andpeptides for 4 min at 23° C.

FIG. 28 displays the FACS cellular uptake of and d-arginine oligomersand N-hxg peptoids. Jurkat cells were incubated with varyingconcentrations (6.3 μM shown) of fluorescently labeled peptoids andpeptides for 4 min at 23° C.

FIG. 29 shows the FACS cellular uptake of d-arginine oligomers and N-chgpeptoids. Jurkat cells were incubated with varying concentrations (12.5μM shown) of fluorescently labeled peptoids and peptides for 4 min at23° C.

FIG. 30 shows a general strategy for attaching a delivery-enhancingtransporter to a drug that includes a triazole ring structure.

FIG. 31A and FIG. 31B show synthetic schemes for making conjugates inwhich FK506 is attached to a delivery-enhancing transporter.

FIG. 32 show that short oligomers of arginine, but not lysine,effectively enter Caco-2 cells. Caco-2 cells were incubated with varyingconcentrations with each of the peptides shown, washed, and analyzed byflow cytometry. Uptake could be inhibited with preincubation with sodiumazide, demonstrating that it was energy dependent.

FIG. 33 displays the measured fluorescence of Caco-2 cells exposed tofluorescent taxol or fluorescent, nonreleasable taxol conjugates witheither heptamers (r7) or decamers (r10) of D-arginine.

FIG. 34 demonstrates Caco-2 monolayer integrity by the measurement of astable transepithelial electric resistance of greater than 100 ohm cm²for the duration of the experiments described in this report.

FIG. 35 shows the accumulation of either Fl aca r5, Fl aca r9, LuciferYellow, or hydrocortisone in the basolateral chamber of a diffusionapparatus after transport through a Caco-2 cell monolayer after onehour.

FIG. 36 displays the blood levels of CsA in rats measured by LC MS MS atthirty minute intervals after intracolonic injection. CsA wasadministered in Cremophor El:ethanol, 1:1, whereas the CsA conjugateswere administered in PBS. The half life in PBS, pH 7.4 at 37° C., of theCsA conjugate (CGC1072) was 1.5 hours.

FIG. 37 displays the blood levels of taxol in rats measured by LC MS MSat thirty minute intervals after intracolonic injection. Taxol wasadministered in Cremophor EL:ethanol, 1:1, whereas the two taxolconjugates were administered in PBS. The half life in PBS, pH 7.4 at 37°C., of the slower releasing conjugate (14) was 5 hours, while that ofthe faster conjugate (13) was ten minutes. See, Example 18.

FIG. 38 displays the blood levels of taxol in rats measured by LC MS MSat thirty minute intervals after buccal delivery. Taxol was administeredin Cremophor El:ethanol, 1:1, whereas the taxol conjugate wasadministered in PBS. The half life in PBS, pH 7.4 at 37° C., of theconjugate (13) was ten minutes.

FIG. 39 illustrates the conjugation of acyclovir to r7-amide via anN-terminal cysteine group. Conjugation with a biotin-containingtransporter is also shown.

FIG. 40 illustrates the conjugate formed between a retinal and a r9(shown without spacing amino acids).

FIG. 41 illustrates the use of a cleavable linker in preparing aretinoic acid-r9 conjugate.

DETAILED DESCRIPTION Definitions

An “epithelial tissue” is the basic tissue that covers surface areas ofthe surface, spaces, and cavities of the body. Epithelial tissues arecomposed primarily of epithelial cells that are attached to one anotherand rest on an extracellular matrix (basement membrane) that istypically produced by the cells. Epithelial tissues include threegeneral types based on cell shape: squamous, cuboidal, and columnarepithelium. Squamous epithelium, which lines lungs and blood vessels, ismade up of flat cells. Cuboidal epithelium lines kidney tubules and iscomposed of cube shaped cells, while columnar epithelium cells line thedigestive tract and have a columnar appearance. Epithelial tissues canalso be classified based on the number of cell layers in the tissue. Forexample, a simple epithelial tissue is composed of a single layer ofcells, each of which sits on the basement membrane. A “stratified”epithelial tissue is composed of several cells stacked upon one another;not all cells contact the basement membrane. A “pseudostratified”epithelial tissue has cells that, although all contact the basementmembrane, appear to be stratified because the nuclei are at variouslevels.

The term “trans-epithelial” delivery or administration refers to thedelivery or administration of agents by permeation through one or morelayers of a body surface or tissue, such as intact skin or a mucousmembrane, by topical administration. Thus, the term is intended toinclude both transdermal (e.g., percutaneous adsorption) andtransmucosal administration. Delivery can be to a deeper layer of thetissue, for example, and/or delivery to the bloodstream.

“Delivery enhancement, “penetration enhancement” or “permeationenhancement” as used herein relates to an increase in amount and/or rateof delivery of a compound that is delivered into and across one or morelayers of an epithelial or endothelial tissue. An enhancement ofdelivery can be observed by measuring the rate and/or amount of thecompound that passes through one or more layers of animal or human skinor other tissue. Delivery enhancement also can involve an increase inthe depth into the tissue to which the compound is delivered, and/or theextent of delivery to one or more cell types of the epithelial or othertissue (e.g., increased delivery to fibroblasts, immune cells, andendothelial cells of the skin or other tissue). Such measurements arereadily obtained by, for example, using a diffusion cell apparatus asdescribed in U.S. Pat. No. 5,891,462.

The amount or rate of delivery of an agent across and/or into skin orother epithelial or endothelial membrane is sometimes quantitated interms of the amount of compound passing through a predetermined area ofskin or other tissue, which is a defined area of intact unbroken livingskin or mucosal tissue. That area will usually be in the range of about5 cm² to about 100 cm², more usually in the range of about 10 cm² toabout 100 cm², still more usually in the range of about 20 cm² to about60 cm².

The terms “guanidyl,” guanidinyl” and “guanidino” are usedinterchangeably to refer to a moiety having the formula —HN═C(NH₂)NH(unprotonated form). As an example, arginine contains a guanidyl(guanidino) moiety, and is also referred to as2-amino-5-guanidinovaleric acid or α-amino-δ-guanidinovaleric acid.“Guanidium” refers to the positively charged conjugate acid form. Theterm “guanidino moiety” includes, for example, guanidine, guanidinium,guanidine derivatives such as (RNHC(NH)NHR′), monosubstitutedguanidines, monoguanides, biguanides, biguanide derivatives such as(RNHC(NH)NHC(NH)NHR′), and the like. In addition, the term “guanidinomoiety” encompasses any one or more of a guanide alone or a combinationof different guanides.

“Amidinyl” and “amidino” refer to a moiety having the formula—C(═NH)(NH₂). “Amidinium” refers to the positively charged conjugateacid form.

The term “trans-barrier concentration” or “trans-tissue concentration”refers to the concentration of a compound present on the side of one ormore layers of an epithelial or endothelial barrier tissue that isopposite or “trans” to the side of the tissue to which a particularcomposition has been added. For example, when a compound is applied tothe skin, the amount of the compound measured subsequently across one ormore layers of the skin is the trans-barrier concentration of thecompound.

“Biologically active agent” or “biologically active substance” refers toa chemical substance, such as a small molecule, macromolecule, or metalion, that causes an observable change in the structure, function, orcomposition of a cell upon uptake by the cell. Observable changesinclude increased or decreased expression of one or more mRNAs,increased or decreased expression of one or more proteins,phosphorylation of a protein or other cell component, inhibition oractivation of an enzyme, inhibition or activation of binding betweenmembers of a binding pair, an increased or decreased rate of synthesisof a metabolite, increased or decreased cell proliferation, and thelike.

The terms “therapeutic agent”, “therapeutic composition”, and“therapeutic substance” refer, without limitation, to any compositionthat can be used to the benefit of a mammalian species. Such agents maytake the form of ions, small organic molecules, peptides, proteins orpolypeptides, oligonucleotides, and oligosaccharides, for example.

The term “macromolecule” as used herein refers to large molecules (MWgreater than 1000 daltons) exemplified by, but not limited to, peptides,proteins, oligonucleotides and polynucleotides of biological orsynthetic origin.

“Small organic molecule” refers to a carbon-containing agent having amolecular weight (MW) of less than or equal to 1000 daltons.

The terms “non-polypeptide agent” and “non-polypeptide therapeuticagent” refer to the portion of a conjugate that does not include thedelivery-enhancing transporter, and that is a biologically active agentother than a polypeptide. An example of a non-polypeptide agent is ananti-sense oligonucleotide, which can be conjugated to a poly-argininepeptide to form a conjugate for enhanced delivery into and across one ormore layers of an epithelial or endothelial tissue.

A “subunit,” as used herein, is a monomeric unit that are joined to forma larger polymeric compound. The set of amino acids are an example ofsubunits. Each amino acid shares a common backbone (—C—C—N—), and thedifferent amino acids differ in their sidechains. The backbone isrepeated in a polypeptide. A subunit represents the shortest repeatingpattern of elements in a polymer backbone. For example, two amino acidsof a peptide are not considered a peptide because two amino acids wouldnot have the shortest repeating pattern of elements in the polymerbackbone.

The term “polymer” refers to a linear chain of two or more identical ornon-identical subunits joined by covalent bonds peptide is an example ofa polymer; peptides can be composed of identical or non-identical aminoacid subunits that are joined by peptide linkages (amide bonds).

The term “peptide” as used herein refers to a compound made up of asingle chain of D- or L-amino acids or a mixture of D- and L-amino acidsjoined by peptide bonds. Generally, peptides contain at least two aminoacid residues and are less than about 50 amino acids in length. D-aminoacids are represented herein by a lower-case one-letter amino acidsymbol (e.g., r for D-arginine), whereas L-amino acids are representedby an upper case one-letter amino acid symbol (e.g., R for L-arginine).Homopolymer peptides are represented by a one-letter amino acid symbolfollowed by the number of consecutive occurrences of that amino acid inthe peptide—(e.g., R7 represents a heptamer that consists of L-arginineresidues).

The term “protein” as used herein refers to a compound that is composedof linearly arranged amino acids linked by peptide bonds, but incontrast to peptides, has a well-defined conformation. Proteins, asopposed to peptides, generally consist of chains of 50 or more aminoacids.

“Polypeptide” as used herein refers to a polymer of at least two aminoacid residues and which contains one or more peptide bonds.“Polypeptide” encompasses pep-tides and proteins, regardless of whetherthe polypeptide has a well-defined conformation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides compositions and methods that enhance thetransfer of compounds, including drugs and other biologically activecompounds, into and across one or more layers of an animal epithelial orendothelial tissue. The methods involve contacting the tissue with aconjugate that includes the compound of interest linked to adelivery-enhancing transporter. The delivery enhancing transportersprovided by the invention are molecules that include sufficientguanidino or amidino moieties to increase delivery of the conjugate intoand across one or more intact epithelial and endothelial tissue layers.The methods and compositions are useful for trans-epithelial andtrans-endothelial delivery of drugs and other biologically activemolecules, and also for delivery of imaging and diagnostic molecules.The methods and compositions of the invention are particularly usefulfor delivery of compounds that require trans-epithelial ortrans-endothelial transport to exhibit their biological effects, andthat by themselves (without conjugation to a delivery-enhancingtransporters or some other modification), are unable, or only poorlyable, to cross such tissues and thus exhibit biological activity.

The delivery-enhancing transporters and methods of the invention providesignificant advantages over previously available methods for obtainingtrans-epithelial and trans-endothelial tissue delivery of compounds ofinterest. The transporters make possible the delivery of drugs and otheragents across tissues that were previously impenetrable to the drug. Forexample, while delivery of drugs across skin was previously nearlyimpossible for all but a few compounds, the methods of the invention candeliver compounds not only into cells of a first layer of an epithelialtissue such as skin, but also across one or more layers of the skin. Theblood brain barrier is also resistant to transport of drugs and otherdiagnostic and therapeutic reagents; the methods and transporters of theinvention provide means to obtain such transport.

The delivery-enhancing transporters increase delivery of the conjugateinto and across one or more intact epithelial or endothelial tissuelayers compared to delivery of the compound in the absence of thedelivery-enhancing transporter. The delivery-enhancing transporters can,in some embodiments, increase delivery of the conjugate significantlyover that obtained using the tat protein of HIV-1 (Frankel et al. (1991)PCT Pub. No. WO 91/09958). Delivery is also increased significantly overthe use of shorter fragments of the tat protein containing the tat basicregion (residues 49-57 having the sequence RKKRRQRRR) (Barsoum et al.(1994) WO 94/04686 and Fawell et al. (1994) Proc. Nat'l. Acad. Sci. USA91: 664-668). Preferably, delivery obtained using the transporters ofthe invention is increased more than 2-fold, still more preferablysix-fold, still more preferably ten-fold, and still more preferablytwenty-fold, over that obtained with tat residues 49-57. In someembodiments, the compositions of the invention do not include tatresidues 49-57.

Similarly, the delivery-enhancing transporters of the invention canprovide increased delivery compared to a 16 amino acidpeptide-cholesterol conjugate derived from the Antennapedia homeodomainthat is rapidly internalized by cultured neurons (Brugidou et al. (1995)Biochem. Biophys. Res. Commun. 214: 685-93). This region, residues 43-58at minimum, has the amino acid sequence RQIKIWFQNRRMKWKK. The Herpessimplex protein VP22, like tat and the Antennapedia domain, waspreviously known to enhance transport into cells, but was not known toenhance transport into and across endothelial and epithelial membranes(Elliot and O'Hare (1997) Cell 88: 223-33; Dilber et al. (1999) GeneTher. 6: 12-21; Phelan et al. (1998) Nature Biotechnol. 16: 440-3). Inpresently preferred embodiments, the delivery-enhancing transportersprovide significantly increased delivery compared to the Antennapediahomeodomain and to the VP22 protein. In some embodiments, thecompositions of the invention do not include the Antennapediahomeodomain, the VP22 protein or eight contiguous arginines.

Structure of Delivery-Enhancing Transporters

The delivery-enhancing transporters of the invention are molecules thathave sufficient guanidino and/or amidino moieties to increase deliveryof a compound to which the delivery-enhancing transporter is attachedinto and across one or more layers of an epithelial tissue (e.g., skinor mucous membrane) or an endothelial tissue (e.g., the blood-brainbarrier). The delivery-enhancing transporters generally include abackbone structure to which is attached the guanidino and/or amidinosidechain moieties. In some embodiments, the backbone is a polymer thatconsists of subunits (e.g., repeating monomer units), at least some ofwhich subunits contain a guanidino or amidino moiety.

A. Guanidino and/or Amidino Moieties

The delivery-enhancing transporters typically display at least 5guanidino and/or amidino moieties, and more preferably 7 or more suchmoieties. Preferably, the delivery-enhancing transporters have 25 orfewer guanidino and/or amidino moieties, and often have 15 or fewer ofsuch moieties. In some embodiments, the delivery-enhancing transporterconsists essentially of 50 or fewer subunits, and can consistessentially of 25 or fewer, 20 or fewer, or 15 or fewer subunits. Thedelivery-enhancing transporter can be as short as 5 subunits, in whichcase all subunits include a guanidino or amidino sidechain moiety. Thedelivery-enhancing transporters can have, for example, at least 6subunits, and in some embodiments have at least 7 or 10 subunits.Generally, at least 50% of the subunits contain a guanidino or amidinosidechain moiety. More preferably, at least 70% of the subunits, andsometimes at least 90% of the subunits in the delivery-enhancingtransporter contain a guanidino or amidino sidechain moiety.

Some or all of the guanidino and/or amidino moieties in thedelivery-enhancing transporters can be contiguous. For example, thedelivery-enhancing transporters can include from 6 to 25 contiguousguanidino and/or amidino-containing subunits. Seven or more contiguousguanidino and/or amidino-containing subunits are present in someembodiments. In some embodiments, each subunit that contains a guanidinomoiety is contiguous, as exemplified by a polymer containing at leastsix contiguous arginine residues.

The delivery-enhancing transporters are exemplified by peptides.Arginine residues or analogs of arginine can constitute the subunitsthat have a guanidino moiety. Such an arginine-containing peptide can becomposed of either all D-, all L- or mixed D- and L-amino acids, and caninclude additional amino acids, amino acid analogs, or other moleculesbetween the arginine residues. Optionally, the delivery-enhancingtransporter can also include a non-arginine residue to which a compoundto be delivered is attached, either directly or through a linker. Theuse of at least one D-arginine in the delivery-enhancing transporterscan enhance biological stability of the transporter during transit ofthe conjugate to its biological target. In some cases thedelivery-enhancing transporters are at least about 50% D-arginineresidues, and for even greater stability transporters in which all ofthe subunits are D-arginine residues are used. If the delivery enhancingtransporter molecule is a peptide, the transporter is not attached to anamino acid sequence to which the amino acids that make up the deliveryenhancing transporter molecule are attached in a naturally occurringprotein.

Preferably, the delivery-enhancing transporter is linear. In a preferredembodiment, an agent to be delivered into and across one or more layersof an epithelial tissue is attached to a terminal end of thedelivery-enhancing transporter. In some embodiments, the agent is linkedto a single transport polymer to form a conjugate. In other embodiments,the conjugate can include more than one delivery-enhancing transporterlinked to an agent, or multiple agents linked to a singledelivery-enhancing transporter.

More generally, it is preferred that each subunit contains a highlybasic sidechain moiety which (i) has a pKa of greater than 11, morepreferably 12.5 or greater, and (ii) contains, in its protonated state,at least two geminal amino groups (NH₂) which share aresonance-stabilized positive charge, which gives the moiety a bidentatecharacter.

The guanidino or amidino moieties extend away from the backbone byvirtue of being linked to the backbone by a sidechain linker. Thesidechain atoms are preferably provided as methylene carbon atoms,although one or more other atoms such as oxygen, sulfur or nitrogen canalso be present. For example, a linker that attaches a guanidino moietyto a backbone can be shown as:

In these formulae, n is preferably at least 2, and is preferably between2 and 7. In some embodiments, n is 3 (arginine for structure 1). Inother embodiments, n is between 4 and 6; most preferably n is 5 or 6.Although the sidechain in the exemplified formulae is shown as beingattached to a peptide backbone (i.e., a repeating amide to which thesidechain is attached to the carbon atom that is a to the carbonylgroup, subunit 1) and a peptoid backbone (i.e., a repeating amide towhich the sidechain is attached to the nitrogen atom that is β to thecarbonyl group, subunit 2), other non-peptide backbones are alsosuitable, as discussed in more detail herein. Thus, similar sidechainlinkers can be attached to nonpeptide backbones (e.g., peptoidbackbones).

In some embodiments, the delivery-enhancing transporters are composed oflinked subunits, at least some of which include a guanidino and/oramidino moiety. Examples of suitable subunits having guanidino and/oramidino moieties are described below.

Amino Acids.

In some embodiments, the delivery-enhancing transporters are composed ofD or L amino acid residues. The amino acids can be naturally occurringor non-naturally occurring amino acids. Arginine(α-amino-δ-guanidinovaleric acid) and α-amino-ε-amidino-hexanoic acid(isosteric amidino analog) are examples of suitable guanidino- andamidino-containing amino acid subunits. The guanidinium group inarginine has a pKa of about 12.5. In some preferred embodiments thetransporters are comprised of at least six contiguous arginine residues.

Other amino acids, such as α-amino-β-guanidino-propionic acid,α-amino-γ-guanidino-butyric acid, or α-amino-ε-guanidino-caproic acid(containing 2, 3 or 5 sidechain linker atoms, respectively, between thebackbone chain and the central guanidinium carbon) can also be used.

D-amino acids can also be used in the delivery enhancing transporters.Compositions containing exclusively D-amino acids have the advantage ofdecreased enzymatic degradation. However, they can also remain largelyintact within the target cell. Such stability is generally notproblematic if the agent is biologically active when the polymer isstill attached. For agents that are inactive in conjugate form, a linkerthat is cleavable at the site of action (e.g., by enzyme- orsolvent-mediated cleavage within a cell) should be included within theconjugate to promote release of the agent in cells or organelles.

In addition, the transport moieties are amino acid oligomers of thefollowing formulae: (ZYZ)_(n)Z, (ZY)_(n)Z, (ZYY)_(n)Z and (ZYYY)_(n)Z.See, U.S. Patent Application No. 60/269,627 filed Feb. 16, 2001. “Z” inthe formulae is D or L-arginine. “Y” is an amino acid that does notcontain a guanidyl or amidinyl moiety. The subscript “n” is an integerranging from 2 to 25.

In the above transport moiety formulae, the letter “Y” represents anatural or non-natural amino acid. The amino acid can be essentially anycompound having (prior to incorporation into the transport moiety) anamino group (NH₂ or NH-alkyl) and a carboxylic acid group (CO₂H) and notcontaining either a guanidyl or amidinyl moiety. Examples of suchcompounds include D and L-alanine, D and L-cysteine, D and L-asparticacid, D and L-glutamic acid, D and L-phenylalanine, glycine, D andL-histidine, D and L-isoleucine, D and L-lysine, D and L-leucine, D andL-methionine, D and L-asparagine, D and L-proline, D and L-glutamine, Dand L-serine, D and L-threonine, D and L-valine, D and L-tryptophan, Dand L-hydroxyproline, D and L-tyrosine, sarcosine, β-alanine, γ-aminobutyric acid and s-amino caproic acid. In each of the above formulae,each Y will be independent of any other Y present in the transportmoiety, though in some embodiments, all Y groups can be the same.

In one group of preferred embodiments, the transport moiety has theformula (ZYZ)_(n)Z, wherein each “Y” is independently selected fromglycine, β-alanine, γ-amino butyric acid and ε-amino caproic acid, “Z”is preferably L-arginine, and n is preferably an integer ranging from 2to 5. More preferably, each “Y” is glycine or s-amino caproic acid and nis 3. Within this group of embodiments, the use of glycine is preferredfor those compositions in which the transport moiety is fused orcovalently attached directly to a polypeptide biological agent such thatthe entire composition can be prepared by recombinant methods. For thoseembodiments in which the transport moiety is to be assembled using, forexample, solid phase methods, s-amino caproic acid is preferred.

In another group of preferred embodiments, the transport moiety has theformula (ZY)_(n)Z, wherein each “Y” is preferably selected from glycine,β-alanine, γ-amino butyric acid and ε-amino caproic acid, “Z” ispreferably L-arginine, and n is preferably an integer ranging from 4 to10. More preferably, each “Y” is glycine or E-amino caproic acid and nis 6. As with the above group of specific embodiments, the use ofglycine is preferred for those compositions in which the transportmoiety is fused or covalently attached directly to a polypeptidebiological agent such that the entire composition can be prepared byrecombinant methods. For solution or solid phase construction of thetransport moiety, ε-amino caproic acid is preferred.

In yet another group of preferred embodiments, the transport moiety hasthe formula (ZYY)_(n)Z, wherein each “Y” is preferably selected fromglycine, β-alanine, γ-amino butyric acid and ε-amino caproic acid, “Z”is preferably L-arginine, and n is preferably an integer ranging from 4to 10. More preferably, each “Y” is glycine or ε-amino caproic acid andn is 6.

In still another group of preferred embodiments, the transport moietyhas the formula (ZYYY)_(n)Z, wherein each “Y” is preferably selectedfrom glycine, β-alanine, γ-amino butyric acid and ε-amino caproic acid,“Z” is preferably L-arginine, and n is preferably an integer rangingfrom 4 to 10. More preferably, “Y” is glycine and n is 6.

In other embodiments, each of the Y groups will be selected to enhancecertain desired properties of the transport moeity. For example, whentransport moeities having a more hydrophobic character are desired, eachY can be selected from those naturally occurring amino acids that aretypically grouped together as hydrophobic amino acids (e.g.,phenylalanine, phenylglycine, valine, leucine, isoleucine). Similarly,transport moieties having a more hydrophilic character can be preparedwhen some or all of the Y groups are hydrophilic amino acids (e.g.,lysine, serine, threonine, glutamic acid, and the like).

One of skill in the art will appreciate that the transport moiety can bea polypeptide fragment within a larger polypeptide. For example, thetransport moiety can be of the formula (ZYY)_(n)Z yet have additionalamino acids which flank this moiety (e.g., X_(m)(ZYY)_(n)Z—X_(p) whereinthe subscripts m and p represent integers of zero to about 10 and each Xis independently a natural or non-natural amino acid).

Other Subunits.

Subunits other than amino acids can also be selected for use in formingtransport polymers. Such subunits can include, but are not limited to,hydroxy amino acids, N-methyl-amino acids amino aldehydes, and the like,which result in polymers with reduced peptide bonds. Other subunit typescan be used, depending on the nature of the selected backbone, asdiscussed in the next section.

B. Backbones

The guanidino and/or amidino moieties that are included in thedelivery-enhancing transporters are generally attached to a linearbackbone. The backbone can comprise a variety of atom types, includingcarbon, nitrogen, oxygen, sulfur and phosphorus, with the majority ofthe backbone chain atoms typically consisting of carbon. A plurality ofsidechain moieties that include a terminal guanidino or amidino groupare attached to the backbone. Although spacing between adjacentsidechain moieties is typically consistent, the delivery-enhancingtransporters used in the invention can also include variable spacingbetween sidechain moieties along the backbone.

A more detailed backbone list includes N-substituted amide (CONRreplaces CONH linkages), esters (CO₂), keto-methylene (COCH₂) reduced ormethyleneamino (CH₂NH), thioamide (CSNH), phosphinate (PO₂RCH₂),phosphonamidate and phosphonamidate ester (PO₂RNH), retropeptide (NHCO),trans-alkene (CR═CH), fluoroallcene (CF═CH), dimethylene (CH₂CH₂),thioether (CH₂S), hydroxyethylene (CH(OH)CH₂), methyleneoxy (CH₂O),tetrazole (CN₄), retrothioamide (NHCS), retroreduced (NHCH₂),sulfonamido (SO₂NH), methylenesulfonamido (CHRSO₂NH), retrosulfonamide(NHSO₂), and peptoids (N-substituted amides), and backbones withmalonate and/or gem-diamino-alkyl subunits, for example, as reviewed byFletcher et al. ((1998) Chem. Rev. 98:763) and detailed by referencescited therein. Many of the foregoing substitutions result inapproximately isosteric polymer backbones relative to backbones formedfrom α-amino acids.

As mentioned above, in a peptoid backbone, the sidechain is attached tothe backbone nitrogen atoms rather than the carbon atoms. (See e.g.,Kessler (1993) Angew. Chem. Int. Ed. Engl. 32:543; Zuckerman et al.(1992) Chemtracts-MacrornoL Chem. 4:80; and Simon et al. (1992) Proc.Nat'l. Acad. Sci. USA 89:9367.) An example of a suitable peptoidbackbone is poly-(N-substituted)glycine (poly-NSG). Synthesis ofpeptoids is described in, for example, U.S. Pat. No. 5,877,278. As theterm is used herein, transporters that have a peptoid backbone areconsidered “non-peptide” transporters, because the transporters are notcomposed of amino acids having naturally occurring sidechain locations.Non-peptide backbones, including peptoid backbones, provide enhancedbiological stability (for example, resistance to enzymatic degradationin vivo).

C. Synthesis of Delivery-enhancing Transporters

Delivery-enhancing transporters are constructed by any method known inthe art. Exemplary peptide polymers can be produced synthetically,preferably using a peptide synthesizer (e.g., an Applied BiosystemsModel 433) or can be synthesized recombinantly by methods well known inthe art. Recombinant synthesis is generally used when the deliveryenhancing transporter is a peptide which is fused to a polypeptide orprotein of interest.

N-methyl and hydroxy-amino acids can be substituted for conventionalamino acids in solid phase peptide synthesis. However, production ofdelivery-enhancing transporters with reduced peptide bonds requiressynthesis of the dimer of amino acids containing the reduced peptidebond. Such dimers are incorporated into polymers using standard solidphase synthesis procedures. Other synthesis procedures are well knownand can be found, for example, in Fletcher et al. (1998) Chem. Rev.98:763, Simon et al. (1992) Proc. Nat'l. Acad. Sci. USA 89:9367, andreferences cited therein.

The delivery-enhancing transporters of the invention can be flanked byone or more non-guanidino/non-amidino subunits (such as glycine,alanine, and cysteine, for example), or a linker (such as anaminocaproic acid group), that do not significantly affect the rate oftrans-tissue layer transport of the corresponding delivery-enhancingtransporter-containing conjugates. Also, any free amino terminal groupcan be capped with a blocking group, such as an acetyl or benzyl group,to prevent ubiquitination in vivo.

Where the transporter is a peptoid polymer, one synthetic methodinvolves the following steps: 1) a peptoid polyamine is treated with abase and pyrazole-1-carboxamidine to provide a mixture; 2) the mixtureis heated and then allowed to cool; 3) the cooled mixture is acidified;and 4) the acidified mixture is purified. Preferably the base used instep 1 is a carbonate, such as sodium carbonate, and heating step 2involves heating the mixture to approximately 50° C. for between about24 hours and about 48 hours. The purification step preferably involveschromatography (e.g., reverse-phase HPLC).

D. Attachment of Transport Polymers to Biologically Active Agents

The agent to be transported can be linked to the delivery-enhancingtransporter according to a number of embodiments. In one embodiment, theagent is linked to a single delivery-enhancing transporter, either vialinkage to a terminal end of the delivery-enhancing transporter or to aninternal subunit within the reagent via a suitable linking group.

In a second embodiment, the agent is attached to more than onedelivery-enhancing transporter, in the same manner as above. Thisembodiment is somewhat less preferred, since it can lead to crosslinkingof adjacent cells.

In a third embodiment, the conjugate contains two agent moietiesattached to each terminal end of the delivery-enhancing transporter. Forthis embodiment, it is presently preferred that the agent has amolecular weight of less than 10 kDa.

With regard to the first and third embodiments just mentioned, the agentis generally not attached to one any of the guanidino or amidinosidechains so that they are free to interact with the target membrane.

The conjugates of the invention can be prepared by straightforwardsynthetic schemes. Furthermore, the conjugate products are usuallysubstantially homogeneous in length and composition, so that theyprovide greater consistency and reproducibility in their effects thanheterogeneous mixtures.

According to an important aspect of the present invention, it has beenfound by the applicants that attachment of a single delivery-enhancingtransporter to any of a variety of types of biologically active agentsis sufficient to substantially enhance the rate of uptake of an agentinto and across one or more layers of epithelial and endothelialtissues, even without requiring the presence of a large hydrophobicmoiety in the conjugate. In fact, attaching a large hydrophobic moietycan significantly impede or prevent cross-layer transport due toadhesion of the hydrophobic moiety to the lipid bilayer of cells thatmake up the epithelial or endothelial tissue. Accordingly, the presentinvention includes conjugates that do not contain substantiallyhydrophobic moieties, such as lipid and fatty acid molecules.

Delivery-enhancing transporters of the invention can be attachedcovalently to biologically active agents by chemical or recombinantmethods.

1. Chemical Linkages

Biologically active agents such as small organic molecules andmacromolecules can be linked to delivery-enhancing transporters of theinvention via a number of methods known in the art (see, for example,Wong, S. S., Ed., Chemistry of Protein Conjugation and Cross-Linking,CRC Press, Inc., Boca Raton, Fla. (1991), either directly (e.g., with acarbodiimide) or via a linking moiety. In particular, carbamate, ester,thioether, disulfide, and hydrazone linkages are generally easy to formand suitable for most applications. Ester and disulfide linkages arepreferred if the linkage is to be readily degraded in the cytosol, aftertransport of the substance across the cell membrane.

Various functional groups (hydroxyl, amino, halogen, etc.) can be usedto attach the biologically active agent to the transport polymer. Groupswhich are not known to be part of an active site of the biologicallyactive agent are preferred, particularly if the polypeptide or anyportion thereof is to remain attached to the substance after delivery.

Polymers, such as peptides produced as described in PCT applicationUS98/10571 (Publication No. WO 9852614), are generally produced with anamino terminal protecting group, such as FMOC. For biologically activeagents which can survive the conditions used to cleave the polypeptidefrom the synthesis resin and deprotect the sidechains, the FMOC may becleaved from the N-terminus of the completed resin-bound polypeptide sothat the agent can be linked to the free N-terminal amine. In suchcases, the agent to be attached is typically activated by methods wellknown in the art to produce an active ester or active carbonate moietyeffective to form an amide or carbamate linkage, respectively, with thepolymer amino group. Of course, other linking chemistries can also beused.

To help minimize side-reactions, guanidino and amidino moieties can beblocked using conventional protecting groups, such as carbobenzyloxygroups (CBZ), di-t-BOC, PMC, Pbf, N—NO₂, and the like.

Coupling reactions are performed by known coupling methods in any of anarray of solvents, such as N,N-dimethyl formamide (DMF),N-methylpyrrolidinone, dichloromethane, water, and the like. Exemplarycoupling reagents include, for example, O-benzotriazolyloxytetramethyluronium hexafluorophosphate (HATU), dicyclohexylcarbodiimide, bromo-tris(pyrrolidino) phosphonium bromide (PyBroP), etc.Other reagents can be included, such as N,N-dimethylamino pyridine(DMAP), 4-pyrrolidino pyridine, N-hydroxy succinimide, N-hydroxybenzotriazole, and the like.

2. Fusion Polypeptides

Delivery-enhancing transporters of the invention can be attached tobiologically active polypeptide agents by recombinant means byconstructing vectors for fusion proteins comprising the polypeptide ofinterest and the delivery-enhancing transporter, according to methodswell known in the art. Generally, the delivery-enhancing transportercomponent will be attached at the C-terminus or N-terminus of thepolypeptide of interest, optionally via a short peptide linker.

3. Releasable Linkers

The biologically active agents are, in presently preferred embodiments,attached to the delivery-enhancing transporter using a linkage that isspecifically cleavable or releasable. The use of such linkages isparticularly important for biologically active agents that are inactiveuntil the attached delivery-enhancing transporter is released. In somecases, such conjugates that consist of a drug molecule that is attachedto a delivery-enhancing transporter can be referred to as prodrugs, inthat the release of the delivery-enhancing transporter from the drugresults in conversion of the drug from an inactive to an active form. Asused herein, “cleaved” or “cleavage” of a conjugate or linker refers torelease of a biological agent from a transporter molecule, therebyreleasing an active biological agent. “Specifically cleavable” or“specifically releasable” refers to the linkage between the transporterand the agent being cleaved, rather than the transporter being degraded(e.g., by proteolytic degradation).

In some embodiments, the linkage is a readily cleavable linkage, meaningthat it is susceptible to cleavage under conditions found in vivo. Thus,upon passing into and through one or more layers of an epithelial and/orendothelial tissue, the agent is released from the delivery-enhancingtransporter. Readily cleavable linkages can be, for example, linkagesthat are cleaved by an enzyme having a specific activity (e.g., anesterase, protease, phosphatase, peptidase, and the like) or byhydrolysis. For this purpose, linkers containing carboxylic acid estersand disulfide bonds are sometimes preferred, where the former groups arehydrolyzed enzymatically or chemically, and the latter are severed bydisulfide exchange, e.g., in the presence of glutathione. The linkagecan be selected so it is cleavable by an enzymatic activity that isknown to be present in one or more layers of an epithelial orendothelial tissue. For example, the stratum granulosum of skin has arelatively high concentration of N-peptidase activity.

A specifically cleavable linker can be engineered onto a transportermolecule. For example, amino acids that constitute a proteaserecognition site, or other such specifically recognized enzymaticcleavage site, can be used to link the transporter to the agent.Alternatively, chemical or other types of linkers that are cleavable by,for example, exposure to light or other stimulus can be used to link thetransporter to the agent of interest.

A conjugate in which an agent to be delivered and a delivery-enhancingtransporter are linked by a specifically cleavable or specificallyreleasable linker will have a half-life. The term “half-life” in thiscontext refers to the amount of time required after applying theconjugate to an epithelial or endothelial membrane for one half of theamount of conjugate to become dissociated to release the free agent. Thehalf-life for some embodiments is typically between 5 minutes and 24hours, and more preferably is between 30 minutes and 2 hours. Thehalf-life of a conjugate can be “tuned” or modified, according to theinvention, as described below.

In some embodiments, the cleavage rate of the linkers is pH dependent.For example, a linker can form a stable linkage between an agent and adelivery-enhancing transporter at an acidic pH (e.g., pH 6.5 or less,more preferably about 6 or less, and still more preferably about 5.5 orless). However, when the conjugate is placed at physiological pH (e.g.,pH 7 or greater, preferably about pH 7.4), the linker will undergocleavage to release the agent. Such pH sensitivity can be obtained by,for example, including a functional group that, when protonated (i.e.,at an acidic pH), does not act as a nucleophile. At a higher (e.g.,physiological) pH, the functional group is no longer protonated and thuscan act as a nucleophile. Examples of suitable functional groupsinclude, for example, N and S. One can use such functional groups tofine-tune the pH at which self-cleavage occurs.

In another embodiment, the linking moiety is cleaved throughself-immolation. Such linking moieties in a transportmoiety-biologically active compound conjugate contain a nucleophile(e.g., oxygen, nitrogen and sulfur) distal to the biologically activecompound and a cleavable group (e.g., ester, carbonate, carbamate andthiocarbamate) proximal to the biologically active compound.Intramolecular attack of the nucleophile on the cleavable group resultsin the scission of a covalent bond, thereby releasing the linking moietyfrom the biologically active compound.

Examples of conjugates containing self-immolating linking moieties(e.g., biologically active agent-L-transport moiety conjugates) arerepresented by structures 3, 4 and 5:

wherein: R¹ is the biologically active compound; X is a linkage formedbetween a functional group on the biologically active compound and aterminal functional group on the linking moiety; Y is a linkage formedfrom a functional group on the transport moiety and a functional groupon the linking moiety; A is N or CH; R² is hydrogen, alkyl, aryl,arylalkyl, acyl or allyl; R³ is the transport moiety; R⁴ is S, O, NR⁶ orCR⁷R⁸; R⁵ is H, OH, SH or NHR⁶; R⁶ is hydrogen, alkyl, aryl, acyl orallyl; R⁷ and R⁸ are independently hydrogen or alkyl; k and m areindependently either 1 or 2; and n is an integer ranging from 1 to 10.Non-limiting examples of the X and Y linkages are (in eitherorientation): —C(O)O—, —C(O)NH—, —OC(O)NH—, —S—S—, —C(S)O—, —C(S)NH—,—NHC(O)NH—, —SO₂NH—, —SONH—, phosphate, phosphonate and phosphinate. Oneof skill in the art will appreciate that when the biological agent has ahydroxy functional group, then X will preferably be —OC(O)— or—OC(O)NH—. Similarly, when the linking group is attached to an aminoterminus of the transport moiety, Y will preferably be —C(O)NH—,—NHC(O)NH—, —SO₂NH—, —SONH— or —OC(O)NH— and the like. In each of thegroups provided above, NH is shown for brevity, but each of the linkages(X and Y) can contain substituted (e.g., N-alkyl or N-acyl) linkages aswell.

Turning first to linking groups illustrated by structure 3, an exampleand preferred embodiment is illustrated for formula 3a:

wherein the wavy lines indicate points of attachment to the transportmoiety and to the biologically active compound. Preparation of aconjugate containing this linking group is illustrated in Example 20(FIG. 6). In this Example and FIG. 6, cyclosporin A is treated withchloroacetic anhydride to form the chloroacetate ester 6i (numbering inFIG. 6) which is then combined with benzylamine to form the N-benzylglycine conjugate 6ii. Condensation of the glycine conjugate withBoc-protected diglycine anhydride provides the acid 6iii which isconverted to the more reactive N-hydroxy succinimide ester 6iv and thencombined with the amino terminus of a transport moiety to form an amidelinkage. One of skill in the art will appreciate that the N-benzyl groupcan be replaced with other groups (e.g., alkyl, aryl, allyl and thelike) or that methylene groups can be replaced with, for example,ethylene, propylene and the like. Preferably, the methylene groups areretained as shown in 3a, to provide an appropriate steric or spatialorientation that allows the linkage to be cleaved in vivo (see FIG. 6B).

Accordingly, for structure 3, the following substituents are preferred:A is N; R² is benzyl; k, m and n are 1; X is —OC(O)— and Y is —C(O)NH—.

Linkages of structure 4, are exemplified by formula 4a:

wherein, as above, the wavy lines indicate the point of attachment toeach of the transport moiety and the biologically active agent. Thepreparation of conjugates having linking groups of formula 4a are shownin Examples 20-22. In Example 20 (see scheme in FIG. 39), acyclovir isacylated with α-chloroacetic anhydride to form the α-chloroacetate ester39i. Reaction of 39i with a heptamer of D-arginine having an N-terminalcysteine residue, provides the thioether product 39ii. Alternatively,acyclovir can be attached to the C-terminus of a transport moiety usinga similar linkage formed between acyclovir α-chloroacetate ester and aheptamer of D-arginine having an C-terminal cysteine residue. In thisinstance, the cysteine residue is provided on the r₇ transport moiety asa C-terminal amide and the linkage has the form:

Accordingly, in one group of preferred embodiments, the conjugate isrepresented by formula 5, in which X is —C(O)—; Y is —C(O)NH—; R⁴ is S;R⁵ is NHR⁶; and the subscripts k and m are each 1. In another group ofpreferred embodiments, the conjugate is represented by formula 2, inwhich X is —OC(O)—; Y is —NHC(O)—; R⁴ is S; R⁵ is CONH₂; and thesubscripts k and m are each 1. Particularly preferred conjugates arethose in which R⁶ is hydrogen, methyl, allyl, butyl or phenyl.

Linking groups represented by the conjugates shown in formula 6 aregenerally of the heterobifunctional type (e.g, ε-aminocaproic acid,serine, homoserine, γ-aminobutyric acid, and the like), althoughsuitably protected dicarboxylic acids or diamines are also useful withcertain biological agents.

For structure 6, the following substituents are preferred: R⁵ is NHR⁶,wherein R⁶ is hydrogen, methyl, allyl, butyl or phenyl; k is 2; X is—C(O)O—; and Y is —C(O)NH—.

Self-immolating linkers typically undergo intramolecular cleavage with ahalf-life between about 10 minutes and about 24 hours in water at 37° C.at a pH of approximately 7.4. Preferably, the cleavage half-life isbetween about 20 minutes and about 4 hours in water at 37° C. at a pH ofapproximately 7.4. More preferably, the cleavage half-life is betweenabout 30 minutes and about 2 hours in water at 37° C. at a pH ofapproximately 7.4.

For a conjugate having the structure 3, one can adjust the cleavagehalf-life by varying the R² substituent. By using an R² of increased ordecreased size, one can obtain a conjugate having a longer or shorterhalf-life respectively. R² in structure 3 is preferably methyl, ethyl,propyl, butyl, allyl, benzyl or phenyl.

Where there is a basic or acidic group in a self-immolating linker, onecan oftentimes adjust cleavage half-life according to the pH of theconjugate solution. For instance, the backbone amine group of structure3 is protonated at acidic pH (e.g., pH 5.5).

The amine cannot serve as a nucleophile inducing intramolecular cleavagewhen it is protonated. Upon introduction of the conjugate into a mediumat physiological pH (7.4), however, the amine is unprotonated asignificant portion of the time. The cleavage half-life iscorrespondingly reduced.

In one embodiment, cleavage of a self-immolating linker occurs in twosteps: intramolecular reaction of a nucleophilic group resulting in thecleavage of a portion of the linking moiety; and, elimination of theremaining portion of the linking moiety. The first step of the cleavageis rate-limiting and can be fine-tuned for pH sensitivity and half-life.

Structure 6 is an example of a two-step, self-immolating moiety that isincorporated into a transport moiety-biologically active compoundconjugate:

wherein: R¹ is the biologically active compound; X represents a linkagebetween a functional group on the biologically active compound and afunctional group on the linking moiety; Ar is a substituted orunsubstituted aryl group, wherein the methylene substituent and phenolicoxygen atom are either ortho or para to one another; R³ is the transportmoiety; R⁴ is S, O, NR⁶ or CR⁷R⁸; R⁵ is H, OH, SH or NHR⁶; R⁶ ishydrogen, alkyl, aryl, arylalkyl, acyl or allyl; R⁷ and R⁸ areindependently hydrogen or alkyl; and, k and m are independently either 1or 2.

An example of a suitable linking group to produce a conjugate of formula6 is:

The construction of a conjugate containing a linking group of formula 6ais provided in Example 24 (see also FIG. 14). In this example (andFigure), the α-chloroacetate ester of 2,4-dimethyl-4-hydroxymethylphenol(41i) is coupled to retinoic acid (41ii) using dicyclohexylcarbodiimide(DCC) and 4-dimethylaminopyridine (DMAP) to provide the intermediate41iii. Subsequent coupling of 41iii with a cysteine residue present onthe N-terminus of an arginine heptamer transport moiety provides thetarget conjugate 41iv.

Preferably, the linking groups used in the conjugates of formula 6, arethose in which Ar is an substituted or unsubstituted phenylene group; R⁴is S; R⁵ is NHR⁶, wherein R⁶ is hydrogen, methyl, allyl, butyl, acetylor phenyl; k and m are 1; X is —C(O)O—; and Y is —C(O)O— or —C(O)NH—.More preferably, R⁶ is hydrogen or acetyl.

While linking groups above have been described with reference toconjugates containing arginine heptamers, one of skill in the art willunderstand that the technology is readily adapted to conjugates with the“spaced” arginine transport moieties of the present invention.

Still other useful linking groups for use in the present invention havebeen described in copending PCT applications. See, for example PCTapplications US98/10571 (Publication No. WO 9852614) and US00/23440(Publication No. WO 01/13957) which describe linking groups for similarcompositions, e.g., conjugates of biologically active agents andtransport oligomers. The linking technology described therein can beused in the present compositions in a similar manner.

Thus, in one group of embodiments, the linking moiety contains a firstcleavable group distal to the biologically active compound and a secondcleavable group proximal to the biologically active compound. Cleavageof the first cleavable group yields a nucleophile capable of reactingintramolecularly with the second cleavable group, thereby cleaving thelinking moiety from the biologically active compound. Examples ofmethods by which the first group is cleaved include photo-illuminationand enzyme mediated hydrolysis. This methodology has been illustratedfor various related small molecule conjugates discussed in PCTapplication US98/10571 (Publication No. WO 9852614).

In one approach, the conjugate can include a disulfide linkage, asillustrated in FIG. 5A of PCT application US00/23440 (Publication No. WO01/13957), (see also, PCT application US98/10571 (Publication No. WO9852614)), which shows a conjugate (I) containing a transport polymer Twhich is linked to a cytotoxic agent, 6-mercaptopurine, by anN-acetyl-protected cysteine group which serves as a linker. Thus, thecytotoxic agent is attached by a disulfide bond to the 6-mercapto group,and the transport polymer is bound to the cysteine carbonyl moiety viaan amide linkage. Cleavage of the disulfide bond by reduction ordisulfide exchange results in release of the free cytotoxic agent. Amethod for synthesizing a disulfide-containing conjugate is provided inExample 9A of PCT application US98/10571. The product described thereincontains a heptamer of Arg residues which is linked to 6-mercaptopurineby an N-acetyl-Cys-Ala-Ala linker, where the Ala residues are includedas an additional spacer to render the disulfide more accessible tothiols and reducing agents for cleavage within a cell. The linker inthis example also illustrates the use of amide bonds, which can becleaved enzymatically within a cell.

In another approach, the conjugate includes a photocleavable linker thatis cleaved upon exposure to electromagnetic radiation. Application ofthis methodology is provided for a related system in FIG. 5B of PCTapplication US00/23440 (Publication No. WO 01/13957) which shows aconjugate (II) containing a transport polymer T which is linked to6-mercaptopurine via a meta-nitrobenzoate linking moiety. Polymer T islinked to the nitrobenzoate moiety by an amide linkage to the benzoatecarbonyl group, and the cytotoxic agent is bound via its 6-mercaptogroup to the p-methylene group. The compound can be formed by reacting6-mercaptopurine with p-bromomethyl-m-nitrobenzoic acid in the presenceof NaOCH₃/methanol with heating, followed by coupling of the benzoatecarboxylic acid to a transport polymer, such as the amino group of aγ-aminobutyric acid linker attached to the polymer (see also, e.g.,Example 9B of PCT application US98/10571). Photo-illumination of theconjugate causes release of the 6-mercaptopurine by virtue of the nitrogroup that is ortho to the mercaptomethyl moiety. This approach findsutility in phototherapy methods as are known in the art, particularlyfor localizing drug activation to a selected area of the body.

In one group of preferred embodiments, the cleavable linker containsfirst and second cleavable groups that can cooperate to cleave theoligomer from the biologically active agent, as illustrated by thefollowing approaches. That is, the cleavable linker contains a firstcleavable group that is distal to the agent, and a second cleavablegroup that is proximal to the agent, such that cleavage of the firstcleavable group yields a linker-agent conjugate containing anucleophilic moiety capable of reacting intramolecularly to cleave thesecond cleavable group, thereby releasing the agent from the linker andoligomer.

Reference is again made to co-owned and copending PCT applicationUS00/23440 (Publication No. WO 01/13957), in which FIG. 5C shows aconjugate (III) containing a transport polymer T linked to theanticancer agent, 5-fluorouracil (5FU). In that figure, the linkage isprovided by a modified lysyl residue. The transport polymer is linked tothe α-amino group, and the 5-fluorouracil is linked via the α-carbonyl.The lysyl ε-amino group has been modified to a carbamate ester ofo-hydroxymethyl nitrobenzene, which comprises a first, photolabilecleavable group in the conjugate. Photo-illumination severs thenitrobenzene moiety from the conjugate, leaving a carbamate that alsorapidly decomposes to give the free α-amino group, an effectivenucleophile. Intramolecular reaction of the α-amino group with the amidelinkage to the 5-fluorouracil group leads to cyclization with release ofthe 5-fluorouracil group.

Still other linkers useful in the present invention are provided in PCTapplication US00/23440 (Publication No. WO 01/13957). In particular,FIG. 5D of US00/23440 illustrates a conjugate (IV) containing adelivery-enhancing transporter T linked to 2′-oxygen of the anticanceragent, paclitaxel. The linkage is provided by a linking moiety thatincludes (i) a nitrogen atom attached to the delivery-enhancingtransporter, (ii) a phosphate monoester located para to the nitrogenatom, and (iii) a carboxymethyl group meta to the nitrogen atom, whichis joined to the 2′-oxygen of paclitaxel by a carboxylate ester linkage.Enzymatic cleavage of the phosphate group from the conjugate affords afree phenol hydroxyl group. This nucleophilic group then reactsintramolecularly with the carboxylate ester to release free paclitaxel,fully capable of binding to its biological target. Example 9C of PCTapplication US98/10571 describes a synthetic protocol for preparing iristype of conjugate.

Still other suitable linkers are illustrated in FIG. 5E of PCTapplication US00/23440 (Publication No. WO 01/13957). In the approachprovided therein, a delivery-enhancing transporter is linked to abiologically active agent, e.g., paclitaxel, by an aminoalkyl carboxylicacid. Preferably, the linker amino group is linked to the linkercarboxyl carbon by from 3 to 5 chain atoms (n=3 to 5), preferably either3 or 4 chain atoms, which are preferably provided as methylene carbons.As seen in FIG. 5E, the linker amino group is joined to thedelivery-enhancing transporter by an amide linkage, and is joined to thepaclitaxel moiety by an ester linkage. Enzymatic cleavage of the amidelinkage releases the delivery-enhancing transporter and produces a freenucleophilic amino group. The free amino group can then reactintramolecularly with the ester group to release the linker from thepaclitaxel.

In another approach, the conjugate includes a linker that is labile atone pH but is stable at another pH. For example, FIG. 6 of PCTapplication US00/23440 (Publication No. WO 01/13957) illustrates amethod of synthesizing a conjugate with a linker that is cleaved atphysiological pH but is stable at acidic pH. Preferably, the linker iscleaved in water at a pH of from about 6.6 to about 7.6. Preferably thelinker is stable in water at a pH from about 4.5 to about 6.5.

Uses of Delivery-Enhancing Transporters

The delivery-enhancing transporters find use in therapeutic,prophylactic and diagnostic applications. The delivery-enhancingtransporters can carry a diagnostic or biologically active reagent intoand across one or more layers of skin or other epithelial tissue (e.g.,gastrointestinal, lung, ocular and the like), or across endothelialtissues such as the blood brain barrier. This property makes thereagents useful for treating conditions by delivering agents that mustpenetrate across one or more tissue layers in order to exert theirbiological effect.

Moreover, the transporters of the present invention can also be usedalone, or in combination with another therapeutic or other compound, asa furin inhibitor. For example, in addition to various poly-argininetransporters, the synthetic transporters described herein, includingpeptoid and those transporters comprising non-naturally occurring aminoacids can be used to inhibit furins. See, e.g., Cameron et al., J. Biol.Chem. 275(47): Furins are proteases that convert a variety ofpro-proteins to their active components. Inhibition of furins is useful,for instance, for treating infections by viruses that rely on furinactivity for virulence or replication. See, e.g., Molloy, et al., T.Cell Biol. 9:28-35 (1999).

Similarly, the transporters of the invention are useful inhibitors ofcapthesin C. For example, certain poly arginine compounds are inhibitorsof capthesin C. See, e.g., Horn, et al., Eur. J. Biochem.267(11):3330-3336 (2000). Similarly, the transporters of the invention,including those comprising synthetic amino acids, are useful to inhibitcapthesin C.

In one aspect of the invention, a furin inhibition assay can be used toscreen for additional transporters. For example, candidate transportercompounds can be tested for their ability to compete with poly argininefor their ability to bind furins or capthesin C using standardcompetition assays. Alternatively, candidate transporters can bescreened for their ability to inhibit furin protease activity asdiscussed in Cameron et al., supra. and Horn et al., supra. Particularlyactive candidates can then be further tested for their ability to act astransporters into and/or across tissues such as the epithelium.

Compositions and methods of the present invention have particularutility in the area of human and veterinary therapeutics. Generally,administered dosages will be effective to deliver picomolar tomicromolar concentrations of the therapeutic composition to the effectorsite. Appropriate dosages and concentrations will depend on factors suchas the therapeutic composition or drug, the site of intended delivery,and the route of administration, all of which can be derived empiricallyaccording to methods well known in the art. Further guidance can beobtained from studies using experimental animal models for evaluatingdosage, as are known in the art.

Administration of the compounds of the invention with a suitablepharmaceutical excipient as necessary can be carried out via any of theaccepted Modes of administration. Thus, administration can be, forexample, intravenous, topical, subcutaneous, transcutaneous,intramuscular, oral, intra-joint, parenteral, peritoneal, intranasal, orby inhalation. Suitable sites of administration thus include, but arenot limited to, skin, bronchial, gastrointestinal, anal, vaginal, eye,and ear. The formulations may take the form of solid, semi-solid,lyophilized powder, or liquid dosage forms, such as, for example,tablets, pills, capsules, powders, solutions, suspensions, emulsions,suppositories, retention enemas, creams, ointments, lotions, aerosols,eye drops, or the like, preferably in unit dosage forms suitable forsimple administration of precise dosages.

The compositions typically include a conventional pharmaceutical carrieror excipient and may additionally include other medicinal agents,carriers, adjuvants, and the like. Preferably, the composition will beabout 5% to 75% by weight of a compound or compounds of the invention,with the remainder consisting of suitable pharmaceutical excipients.Appropriate excipients can be tailored to the particular composition androute of administration by methods well known in the art, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co.,Easton, Pa. (1990).

For oral administration, such excipients include pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, andthe like. The composition may take the form of a solution, suspension,tablet, pill, capsule, powder, sustained-release formulation, and thelike.

In some embodiments, the pharmaceutical compositions take the form of apill, tablet or capsule, and thus, the composition can contain, alongwith the biologically active conjugate, any of the following: a diluentsuch as lactose, sucrose, dicalcium phosphate, and the like; adisintegrant such as starch or derivatives thereof; a lubricant such asmagnesium stearate and the like; and a binder such a starch, gum acacia,polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof.

The active compounds of the formulas may be formulated into asuppository comprising, for example, about 0.5% to about 50% of acompound of the invention, disposed in a polyethylene glycol (PEG)carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]). Liquid compositionscan be prepared by dissolving or dispersing compound (about 0.5% toabout 20%), and optional pharmaceutical adjuvants in a carrier, such as,for example, aqueous saline (e.g., 0.9% w/v sodium chloride), aqueousdextrose, glycerol, ethanol and the like, to form a solution orsuspension, e.g., for intravenous administration. The active compoundsmay also be formulated into a retention enema.

If desired, the composition to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, such as, for example, sodium acetate,sorbitan monolaurate, or triethanolamine oleate.

For topical administration, the composition is administered in anysuitable format, such as a lotion or a transdermal patch. For deliveryby inhalation, the composition can be delivered as a dry powder (e.g.,Inhale Therapeutics) or in liquid form via a nebulizer.

Methods for preparing such dosage forms are known or will be apparent tothose skilled in the art; for example, see Remington's PharmaceuticalSciences, supra., and similar publications. The composition to beadministered will, in any event, contain a quantity of the pro-drugand/or active compound(s) in a pharmaceutically effective amount forrelief of the condition being treated when administered in accordancewith the teachings of this invention.

Generally, the compounds of the invention are administered in atherapeutically effective amount, i.e., a dosage sufficient to effecttreatment, which will vary depending on the individual and conditionbeing treated. Typically, a therapeutically effective daily dose is from0.1 to 100 mg/kg of body weight per day of drug. Most conditions respondto administration of a total dosage of between about 1 and about 30mg/kg of body weight per day, or between about 70 mg and 2100 mg per dayfor a 70 kg person.

Stability of the conjugate can be further controlled by the compositionand stereochemistry of the backbone and sidechains of thedelivery-enhancing transporters. For polypeptide delivery-enhancingtransporters, D-isomers are generally resistant to endogenous proteases,and therefore have longer half-lives in serum and within cells.D-polypeptide polymers are therefore appropriate when longer duration ofaction is desired. L-polypeptide polymers have shorter half-lives due totheir susceptibility to proteases, and are therefore chosen to impartshorter acting effects. This allows side-effects to be averted morereadily by withdrawing therapy as soon as side-effects are observed.Polypeptides comprising mixtures of D and L-residues have intermediatestabilities. Homo-D-polymers are generally preferred.

A. Application to Skin

The delivery-enhancing transporters of the invention make possible thedelivery of biologically active and diagnostic agents across the skin.Surprisingly, the transporters can deliver an agent across the stratumcorneum, which previously had been a nearly impenetrable barrier to drugdelivery. The stratum corneum, the outermost layer of the skin, iscomposed of several layers of dead, keratin-filled skin cells that aretightly bound together by a “glue” composed of cholesterol and fattyacids. Once the agents are delivered through the stratum corneum by thetransporters of the invention, the agents can enter the viableepidermis, which is composed of the stratum granulosum, stratum lucidumand stratum germinativum which, along with the stratum corneum, make upthe epidermis. Delivery in some embodiments of the invention is throughthe epidermis and into the dermis, including one or both of thepapillary dermis and the reticular dermis.

This ability to obtain penetration of one or more layers of the skin cangreatly enhance the efficacy of compounds such as antibacterials,antifungals, antivirals, antiproliferatives, immunosuppressives,vitamins, analgesics, hormones, and the like. Numerous such compoundsare known to those of skill in the art (see, e.g., Hardman and Limbird,Goodman & Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill, New York, 1996).

In some embodiments, the agent is delivered into a blood vessel that ispresent in the epithelial tissue, thus providing a means for delivery ofthe agent systemically. Delivery can be either intrafollicular orinterfollicular, or both. Pretreatment of the skin is not required fordelivery of the conjugates.

In other embodiments, the delivery-enhancing transporters are useful fordelivering cosmetics and agents that can treat skin conditions. Targetcells in the skin that are of interest include, for example,fibroblasts, epithelial cells and immune cells. For example, thetransporters provide the ability to deliver compounds such asantiinflammatory agents to immune cells found in the dermis.

Glucocorticoids (adrenocorticoid steroids) are among the compounds forwhich delivery across skin can be enhanced by the delivery-enhancingtransporters of the invention. Conjugated glucocorticoids of theinvention are useful for treating inflammatory skin diseases, forexample. Exemplary glucocorticoids include, e.g., hydrocortisone,prenisone (deltasone) and predrisonlone (hydeltasol). Examples ofparticular conditions include eczema (including atopic dermatitis,contact dermatitis, allergic dermatitis), bullous disease, collagenvascular diseases, sarcoidosis, Sweet's disease, pyoderma gangrenosum,Type I reactive leprosy, capillary hemangiomas, lichen planus,exfoliative dermatitis, erythema nodosum, hormonal abnormalities(including acne and hirsutism), as well as toxic epidermal necrolysis,erythema multiforme, cutaneous T-cell lymphoma, discoid lupuserythematosus, and the like.

Retinoids are another example of a biologically active agent for whichone can use the delivery-enhancing transporters of the invention toenhance delivery into and across one or more layers of the skin or otherepithelial or endothelial tissue. Retinoids that are presently in useinclude, for example retinol, tretinoin, isotretinoin, etretinate,acitretin, and arotinoid. Conditions that are treatable using retinoidsconjugated to the delivery-enhancing transporters of the inventioninclude, but are not limited to, acne, keratinization disorders, skincancer, precancerous conditions, psoriasis, cutaneous aging, discoidlupus erythematosus, scleromyxedema, verrucous epidermal nevus,subcorneal pustular dermatosis, Reiter's syndrome, warts, lichen planus,acanthosis nigricans, sarcoidosis, Grover's disease, porokeratosis, andthe like.

Cytotoxic and immunosuppressive drugs constitute an additional class ofdrugs for which the delivery-enhancing transporters of the invention areuseful. These agents are commonly used to treat hyperproliferativediseases such as psoriasis, as well as for immune diseases such asbullous dermatoses and leukocytoclastic vasculitis. Examples of suchcompounds that one can conjugate to the delivery-enhancing transportersof the invention include, but are not limited to, antimetabolites suchas methotrexate, azathioprine, fluorouracil, hydroxyurea, 6-thioquanine,mycophenolate, chlorambucil, vincristine, vinblastine and dactinomycin.Other examples are alkylating agents such as cyclophosphamide,mechloroethamine hydrochloride, carmustine. taxol, tacrolimus andvinblastine are additional examples of useful biological agents, as aredapsone and sulfasalazine. Immunosuppressive drugs such as cyclosporinand Ascomycins, such as FK506 (tacrolimus), and rapamycin (e.g., U.S.Pat. No. 5,912,253) and analogs of such compounds are of particularinterest (e.g., Mollinson et al., Current Pharm. Design 4(5):367-380(1998); U.S. Pat. Nos. 5,612,350; 5,599,927; 5,604,294; 5,990,131;5,561,140; 5,859,031; 5,925,649; 5,994,299; 6,004,973 and 5,508,397).Cyclosporins include cyclosporin A, B, C, D, G and M. See, e.g., U.S.Pat. Nos. 6,007,840; and 6,004,973. For example, such compounds areuseful in treating psoriasis, eczema (including atopic dermatitis,contact dermatitis, allergic dermatitis) and alopecia greata.

The delivery-enhancing transporters can be conjugated to agents that areuseful for treating conditions such as lupus erythematosus (both discoidand systemic), cutaneous dermatomyositis, porphyria cutanea tarda andpolymorphous light eruption. Agents useful for treating such conditionsinclude, for example, quinine, chloroquine, hydroxychloroquine, andquinacrine.

The delivery-enhancing transporters of the invention are also useful fortransdermal delivery of antiinfective agents. For example,antibacterial, antifungal and antiviral agents can be conjugated to thedelivery-enhancing transporters. Antibacterial agents are useful fortreating conditions such as acne, cutaneous infections, and the like.Antifungal agents can be used to treat tinea corporis, tinea pedis,onychomycosis, candidiasis, tinea versicolor, and the like. Because ofthe delivery-enhancing properties of the conjugates, these conjugatesare useful for treating both localized and widespread infections.Antifungal agents are also useful for treating onychomycosis. Examplesof antifungal agents include, but are not limited to, azole antifungalssuch as itraconazole, myconazole and fluconazole. Examples of antiviralagents include, but are not limited to, acyclovir, famciclovir, andvalacyclovir. Such agents are useful for treating viral diseases, e.g.,herpes.

Another example of a biologically active agent for which enhancement ofdelivery by conjugation to the delivery-enhancing transporters of theinvention is desirable are the antihistamines. These agents are usefulfor treating conditions such as pruritus due to urticaria, atopicdermatitis, contact dermatitis, psoriasis, and many others. Examples ofsuch reagents include, for example, terfenadine, astemizole, lorotadine,cetirizine, acrivastine, temelastine, cimetidine, ranitidine,famotidine, nizatidine, and the like. Tricyclic antidepressants can alsobe delivered using the delivery-enhancing transporters of the invention.

Topical antipsoriasis drugs are also of interest. Agents such ascorticosteroids, calcipotriene, and anthralin can be conjugated to thedelivery-enhancing transporters of the invention and applied to skin.

The delivery-enhancing transporters of the invention are also useful forenhancing delivery of photochemotherapeutic agents into and across oneor more layers of skin and other epithelial tissues. Such compoundsinclude, for example, the psoralens, and the like. Sunscreen componentsare also of interest; these include p-aminobenzoic acid esters,cinnamates and salicylates, as well as benzophenones, anthranilates, andavobenzone.

Pain relief agents and local anesthetics constitute another class ofcompounds for which conjugation to the delivery-enhancing transportersof the invention can enhance treatment. Lidocaine, bupibacaine,novocaine, procaine, tetracaine, benzocaine, cocaine, and the opiates,are among the compounds that one can conjugate to the delivery-enhancingtransporters of the invention. Application of pain relief agents to thejoints or skin near the at the joints, e.g., in patients suffering fromrhematoid arthritis, is also contemplated.

Other biological agents of interest include, for example, minoxidil,keratolytic agents, destructive agents such as podophyllin,hydroquinone, capsaicin, masoprocol, colchicine, and gold.

Treatment of inflammed joints such as occurs in rhematoid arthritis canalso be treated with compounds useful for such treatments conjugated tothe transporters of the invention.

B. Gastrointestinal Administration

The delivery-enhancing transporters of the invention are also useful fordelivery of conjugated drugs by gastrointestinal administration.Gastrointestinal administration can be used for both systemically activedrugs, and for drugs that act in the gastrointestinal epithelium.

Among the gastrointestinal conditions that are treatable usingappropriate reagents conjugated to the delivery-enhancing transportersare inflammatory bowel disease such as Crohn's disease (e.g.,cyclosporin and ascomycins such as FK506; aminosalicylates, e.g.,aspirin-like drugs, which include sulfasalazine and mesalamine;corticosteroids, e.g., prednisone and methylprednisolone; immunemodifiers, e.g., azathioprine, 6 MP, methotrexate; and antibiotics,e.g., metronidazole, ampicillin, ciprofloxacin, and others). Othertreatable gastrointestinal conditions include ulcerative colitis,gastrointestinal ulcers, peptic ulcer disease, imbalance of salt andwater absorption (can lead to constipation, diarrhea, or malnutrition),abnormal proliferative diseases, and the like. Ulcer treatments include,for example, drugs that reduce gastric acid secretion, such as H₂histamine inhibitors (e.g., cymetidine and ranitidine) and inhibitors ofthe proton-potassium ATPase (e.g., lansoprazle and omeprazle), andantibiotics directed at Helicobacter pylori.

Compounds useful for the treatment of constipation can also be used inconjunction with the transporters of the invention. Useful compounds fortreating constipation include, e.g., surfactant laxatives such asdocusate sodium, poloxamer 188, dehydrochloric acid and ricinoleic acid.Exemplary stimulant laxatives include, e.g., phenolphthalein, bisacodyland anthraquinone laxatives such as danthron.

Antibiotics are among the biologically active agents that are usefulwhen conjugated to the delivery-enhancing transporters of the invention,particularly those that act on invasive bacteria, such as Shigella,Salmonella, and Yersinia. Such compounds include, for example,norfloxacin, ciprofloxacin, trimethoprim, sulfamethyloxazole, and thelike.

Anti-neoplastic agents, for example, for the treatment of colon cancer,can also be conjugated to the delivery-enhancing transporters of theinvention and administered by the gastrointestinal route. These include,for example, cisplatin, methotrexate, taxol, fluorouracil,mercaptopurine, donorubicin, bleomycin, streptozocin, mitomycin and thelike.

For gastrointestinal and colonic delivery of orally administeredtransporters or active compounds, it can be beneficial to coat orencapsulate the compounds so that the compounds are not released untilthey are delivered to the gastrointestinal (GD tract or colon. Methodsand composition useful for delivery to the GI tract or colon aredescribed in, e.g., U.S. Pat. Nos. 6,183,466 and 6,120,803.

C. Respiratory Tract Administration

The delivery-enhancing transporters of the invention can also used toenhance administration of drugs through the respiratory tract. Therespiratory tract, which includes the nasal mucosa, hypopharynx, andlarge and small airway structures, provides a large mucosal surface fordrug absorption. The enhanced penetration of the conjugated agents intoand across one or more layers of the epithelial tissue that is providedby the delivery-enhancing transporters of the invention results inamplification of the advantages that respiratory tract delivery has overother delivery methods. For example, lower doses of an agent are oftenneeded to obtain a desired effect, a local therapeutic effect can occurmore rapidly, and systemic therapeutic blood levels of the agent areobtained quickly. Rapid onset of pharmacological activity can resultfrom respiratory tract administration. Moreover, respiratory tractadministration generally has relatively few side effects.

The transporters of the invention can be used to deliver biologicalagents that are useful for treatment of pulmonary conditions. Examplesof conditions treatable by nasal administration include, for example,asthma. These compounds include antiinflammatory agents, such ascorticosteroids, cromolyn, and nedocromil, bronchodialators such asβ2-selective adronergic drugs and theophylline, and immunosuppressivedrugs (e.g., cyclosporin and FK506). Other conditions include, forexample, allergic rhinitis (which can be treated with glucocorticoids),and chronic obstructive pulmonary disease (emphysema). Other drugs thatact on the pulmonary tissues and can be delivered using the transportersof the invention include beta-agonists, mast cell stabilizers,antibiotics, antifungal and antiviral agents, surfactants, vasoactivedrugs, sedatives and hormones.

Respiratory tract administration is useful not only for treatment ofpulmonary conditions, but also for delivery of drugs to distant targetorgans via the circulatory system. A wide variety of such drugs anddiagnostic agents can be administered through the respiratory tractafter conjugation to the delivery-enhancing transporters of theinvention.

D. Delivery of Agents across the Blood Brain Barrier

The delivery-enhancing transporters are also useful for deliveringbiologically active and diagnostic agents across the blood brainbarrier. The agents are useful for treating ischemia (e.g., using ananti-apoptotic drug), as well as for delivering neurotransmitters andother agents for treating various conditions such as schizophrenia,Parkinson's disease, pain (e.g., morphine, the opiates). The5-hydroxytryptamine receptor antagonist is useful for treatingconditions such as migraine headaches and anxiety.

E. Diagnostic Imaging and Contrast Agents

The delivery-enhancing transporters of the invention are also useful fordelivery of diagnostic imaging and contrast agents into and across oneor more layers of an epithelial and/or endothelial tissue. Examples ofdiagnostic agents include substances that are labeled withradioactivity, such as ⁹⁹ mTc glucoheptonate, or substances used inmagnetic resonance imaging (MRI) procedures such as gadolinium dopedchelation agents (e.g. Gd-DTPA). Other examples of diagnostic agentsinclude marker genes that encode proteins that are readily detectablewhen expressed in a cell (including, but not limited to,(β-galactosidase, green fluorescent protein, luciferase, and the like. Awide variety of labels may be employed, such as radionuclides, fluors,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands(particularly haptens), etc.

F. Ocular Administration

The delivery-enhancing transporters of the invention can also used toenhance administration of drugs through the tissues of the eye and otherrelated tissues such as the eye lid. The ocular tissues include thecornea, iris, lens, vitreus, vitreus humor, the optic nerve and theeyelid.

Examples of conditions treatable with the compositions of the inventioninclude the following. Conjunctivitis, sometimes called pink eye, is aninflammation of the blood vessels in the conjunctiva, the membrane thatcovers the sclera and inside of the eyelids. Conjunctivitis may becaused by bacteria or viruses.

Antibacterial and antiviral compounds useful for treating bacterial orviral infections of the eye are well known. See, e.g., Hardman andLimbird, supra. Exemplary compounds include chloramphenicol,Ciproflaxacin, polymyxin B and tetracycline. Exemplary antiviralcompounds include idoxuridine, acyclovir and ganciclovir.

Styes are noncontagious, bacterial infections of one of the sebaceousglands of the eyelid: A stye looks like a small, red bump either on theeyelid or on the edge of the eyelid.

Dry eye, or “Sjögren's syndrome,” is an immune system disordercharacterized by inflammation and dryness of the mouth, eyes, and othermucous membranes, damages the lacrimal glands, and this damage affectstear production. Dry eye can be treated with immunosuppressive compoundssuch as cyclosporin as well as with ascomycins or steroids.

Glaucoma is a condition in which the normal fluid pressure inside theeyes (intraocular pressure, or IOP) slowly rises as a result of thefluid aqueous humor, which normally flows in and out of the eye, notbeing able to drain properly. Instead, the fluid collects and causespressure damage to the optic nerve and loss of vision. Useful compoundsto treat glaucoma, blindness, and other eye disorders include, e.g.,timolol, levobunolol and phenylepherine. Growth factors such as nervegrowth factor (NGF) (see, e.g., Bennett, et al. Mol Ther 1(6):50′-5(2000)) are also useful for treating glaucoma and other oculardisorders.

Therapeutic compounds for treatment of ocular diseases, such as thosediscussed above, are well known to those of skill in the art. Typically,administration of the composition of the invention to the ocular tissuesis in the form of an eye drop. Alternatively, for example, thecompositions can be injected into the eye or applied as an ointment.

Eye drops including the compounds of the invention can also include anisotonic agent added to sterilized purified water, and if required, apreservative, a buffering agent, a stabilizer, a viscous vehicle and thelike are added to the solution and dissolved therein. After dissolution,the pH is adjusted with a pH controller to be within a range suitablefor use as an ophthalmic medicine, preferably within the range of 4.5 to8.

Sodium chloride, glycerin, mannitol or the like may be used as theisotonic agent; p-hydroxybenzoic acid ester, benzalkonium chloride orthe like as the preservative; sodium hydrogenphosphate, sodiumdihydrogenphosphate, boric acid or the like as the buffering agent;sodium edetate or the like as the stabilizer; polyvinyl alcohol,polyvinyl pyrrolidone, polyacrylic acid or the like as the viscousvehicle; and sodium hydroxide, hydrochloric acid or the like as the pHcontroller

Biologically Active and Diagnostic Molecules Useful with theDelivery-Enhancing Transporters

The delivery-enhancing transporters can be conjugated to a wide varietyof biologically active agents and molecules that have diagnostic use.

A. Small Organic Molecules

Small organic molecule therapeutic agents can be advantageously attachedto linear polymeric compositions as described herein, to facilitate orenhance transport across one or more layers of an epithelial orendothelial tissue. For example, delivery of highly charged agents, suchas levodopa (L-3,4-dihydroxy-phenylalanine; L-DOPA) may benefit bylinkage to delivery-enhancing transporters as described herein. Peptoidand peptidomimetic agents are also contemplated (e.g., Langston (1997)DDT 2:255; Giannis et al. (1997) Advances Drug Res. 29:1). Also, theinvention is advantageous for delivering small organic molecules thathave poor solubilities in aqueous liquids, such as serum and aqueoussaline. Thus, compounds whose therapeutic efficacies are limited bytheir low solubilities can be administered in greater dosages accordingto the present invention, and can be more efficacious on a molar basisin conjugate form, relative to the non-conjugate form, due to higheruptake levels by cells.

Since a significant portion of the topological surface of a smallmolecule is often involved, and therefore required, for biologicalactivity, the small molecule portion of the conjugate in particularcases may need to be severed from the attached delivery-enhancingtransporter and linker moiety (if any) for the small molecule agent toexert biological activity after crossing the target epithelial tissue.For such situations, the conjugate preferably includes a cleavablelinker for releasing free drug after passing through an epithelialtissue.

FIG. 5D and FIG. 5E are illustrative of another aspect of the invention,comprising taxane- and taxoid anticancer conjugates which have enhancedtrans-epithelial tissue transport rates relative to correspondingnon-conjugated forms. The conjugates are particularly useful forinhibiting growth of cancer cells. Taxanes and taxoids are believed tomanifest their anticancer effects by promoting polymerization ofmicrotubules (and inhibiting depolymerization) to an extent that isdeleterious to cell function, inhibiting cell replication and ultimatelyleading to cell death.

The term “taxane” refers to paclitaxel (FIG. 5F, R′=acetyl, R″=benzyl)also known under the trademark “TAXOL”) and naturally occurring,synthetic, or bioengineered analogs having a backbone core that containsthe A, B, C and D rings of paclitaxel, as illustrated in FIG. 5G. FIG.5F also indicates the structure of “TAXOTERE™” (R′═H, R″═BOC), which isa somewhat more soluble synthetic analog of paclitaxel sold byRhone-Poulenc. “Taxoid” refers to naturally occurring, synthetic orbioengineered analogs of paclitaxel that contain the basic A, B and Crings of paclitaxel, as shown in FIG. 5H. Substantial synthetic andbiological information is available on syntheses and activities of avariety of taxane and taxoid compounds, as reviewed in Suffness (1995)Taxol: Science and Applications, CRC Press, New York, N.Y., pp. 237-239,particularly in Chapters 12 to 14, as well as in the subsequentpaclitaxel literature. Furthermore, a host of cell lines are availablefor predicting anticancer activities of these compounds against certaincancer types, as described, for example, in Suffness at Chapters 8 and13.

The delivery-enhancing transporter is conjugated to the taxane or taxoidmoiety via any suitable site of attachment in the taxane or taxoid.Conveniently, the transport polymer is linked via a C2′-oxygen atom,C7-oxygen atom, using linking strategies as above. Conjugation of atransport polymer via a C7-oxygen leads to taxane conjugates that haveanticancer and antitumor activity despite conjugation at that position.Accordingly, the linker can be cleavable or non-cleavable. Conjugationvia the C2′-oxygen significantly reduces anticancer activity, so that acleavable linker is preferred for conjugation to this site. Other sitesof attachment can also be used, such as C10.

It will be appreciated that the taxane and taxoid conjugates of theinvention have improved water solubility relative to taxol (≈0.25 μg/mL)and taxotere (6-7 μg/mL). Therefore, large amounts of solubilizingagents such as “CREMOPHOR EL” (polyoxyethylated castor oil), polysorbate80 (polyoxyethylene sorbitan monooleate, also known as “TWEEN 80”), andethanol are not required, so that side-effects typically associated withthese solubilizing agents, such as anaphylaxis, dyspnea, hypotension,and flushing, can be reduced.

B. Metals

Metals can be transported into and across one or more layers ofepithelial and endothelial tissues using chelating agents such astexaphyrin or diethylene triamine pentacetic acid (DTPA), conjugated toa delivery-enhancing transporter of the invention, as illustrated byExample. These conjugates are useful for delivering metal ions forimaging or therapy. Exemplary metal ions include Eu, Lu, Pr, Gd, Tc99m,Ga67, In111, Y90, Cu67, and Co57. Preliminary membrane-transport studieswith conjugate candidates can be performed using cell-based assays suchas described in the Example section below. For example, using europiumions, cellular uptake can be monitored by time-resolved fluorescencemeasurements. For metal ions that are cytotoxic, uptake can be monitoredby cytotoxicity.

C. Macromolecules

The enhanced transport methods of the invention are particularly suitedfor enhancing transport into and across one or more layers of anepithelial or endothelial tissue for a number of macromolecules,including, but not limited to proteins, nucleic acids, polysaccharides,and analogs thereof. Exemplary nucleic acids include oligonucleotidesand polynucleotides formed of DNA and RNA, and analogs thereof, whichhave selected sequences designed for hybridization to complementarytargets (e.g., antisense sequences for single- or double-strandedtargets), or for expressing nucleic acid transcripts or proteins encodedby the sequences. Analogs include charged and preferably unchargedbackbone analogs, such as phosphonates (preferably methyl phosphonates),phosphoramidates (N3′ or N5′), thiophosphates, unchargedmorpholino-based polymers, and protein nucleic acids. (PNAs). Suchmolecules can be used in a variety of therapeutic regimens, includingenzyme replacement therapy, gene therapy, and anti-sense therapy, forexample.

By way of example, protein nucleic acids (PNA) are analogs of DNA inwhich the backbone is structurally homomorphous with a deoxyribosebackbone. The backbone consists of N-(2-aminoethyl)glycine units towhich the nucleobases are attached. PNAs containing all four naturalnucleobases hybridize to complementary oligonucleotides obeyingWatson-Crick base-pairing rules, and is a true DNA mimic in terms ofbase pair recognition (Eghohn et al. (1993) Nature 365:566-568. Thebackbone of a PNA is formed by peptide bonds rather than phosphateesters, making it well-suited for anti-sense applications. Since thebackbone is uncharged, PNA/DNA or PNA/RNA duplexes that form exhibitgreater than normal thermal stability. PNAs have the additionaladvantage that they are not recognized by nucleases or proteases. Inaddition, PNAs can be synthesized on an automated peptides synthesizerusing standard t-Boc chemistry. The PNA is then readily linked to atransport polymer of the invention.

Examples of anti-sense oligonucleotides whose transport into and acrossepithelial and endothelial tissues can be enhanced using the methods ofthe invention are described, for example, in U.S. Pat. No. 5,594,122.Such oligonucleotides are targeted to treat human immunodeficiency virus(HIV). Conjugation of a transport polymer to an anti-senseoligonucleotide can be effected, for example, by forming an amidelinkage between the peptide and the 5′-terminus of the oligonucleotidethrough a succinate linker, according to well-established methods. Theuse of PNA conjugates is further illustrated in Example 11 of PCTApplication PCT/US98/10571. FIG. 7 of that application shows resultsobtained with a conjugate of the invention containing a PNA sequence forinhibiting secretion of gamma-interferon (γ-IFN) by T cells, as detailedin Example 11. As can be seen, the anti-sense PNA conjugate waseffective to block γ-IFN secretion when the conjugate was present atlevels above about 10 μM. In contrast, no inhibition was seen with thesense-PNA conjugate or the non-conjugated antisense PNA alone.

Another class of macromolecules that can be transported across one ormore layers of an epithelial or endothelial tissue is exemplified byproteins, and in particular, enzymes. Therapeutic proteins include, butare not limited to replacement enzymes. Therapeutic enzymes include, butare not limited to, alglucerase, for use in treating lysozomalglucocerebrosidase deficiency (Gaucher's disease), alpha-L-iduronidase,for use in treating mucopolysaccharidosis I,alpha-N-acetylglucosamidase, for use in treating sanfilippo B syndrome,lipase, for use in treating pancreatic insufficiency, adenosinedeaminase, for use in treating severe combined immunodeficiencysyndrome, and triose phosphate isomerase, for use in treatingneuromuscular dysfunction associated with triose phosphate isomerasedeficiency.

In addition, and according to an important aspect of the invention,protein antigens may be delivered to the cytosolic compartment ofantigen-presenting cells (APCs), where they are degraded into peptides.The peptides are then transported into the endoplasmic reticulum, wherethey associate with nascent HLA class I molecules and are displayed onthe cell surface. Such “activated” APCs can serve as inducers of class Irestricted antigen-specific cytotoxic T-lymphocytes (CTLs), which thenproceed to recognize and destroy cells displaying the particularantigen. APCs that are able to carry out this process include, but arenot limited to, certain macrophages, B cells and dendritic cells. In oneembodiment, the protein antigen is a tumor antigen for eliciting orpromoting an immune response against tumor cells. The transport ofisolated or soluble proteins into the cytosol of APC with subsequentactivation of CTL is exceptional, since, with few exceptions, injectionof isolated or soluble proteins does not result either in activation ofAPC or induction of CTLs. Thus, antigens that are conjugated to thetransport enhancing compositions of the present invention may serve tostimulate a cellular immune response in vitro or in vivo.

In another embodiment, the invention is useful for deliveringimmunospecific antibodies or antibody fragments to the cytosol tointerfere with deleterious biological processes such as microbialinfection. Recent experiments have shown that intracellular antibodiescan be effective antiviral agents in plant and mammalian cells (e.g.,Tavladoraki et al. (1993) Nature 366:469; and Shaheen et al. (1996) J.Virol. 70:3392. These methods have typically used single-chain variableregion fragments (scFv), in which the antibody heavy and light chainsare synthesized as a single polypeptide. The variable heavy and lightchains are usually separated by a flexible linker peptide (e.g., of 15amino acids) to yield a 28 kDa molecule that retains the high affinityligand binding site. The principal obstacle to wide application of thistechnology has been efficiency of uptake into infected cells. But byattaching transport polymers to scFv fragments, the degree of cellularuptake can be increased, allowing the immunospecific fragments to bindand disable important microbial components, such as HIV Rev, HIV reversetranscriptase, and integrase proteins.

D. Peptides

Peptides to be delivered by the enhanced transport methods describedherein include, but should not be limited to, effector polypeptides,receptor fragments, and the like. Examples include peptides havingphosphorylation sites used by proteins mediating intra-cellular signals.Examples of such proteins include, but are not limited to, proteinkinase C, RAF-1, p21Ras, NF-κB, C-JUN, and cytoplasmic tails of membranereceptors such as IL-4 receptor, CD28, CTLA-4, V7, and MHC Class I andClass II antigens.

When the delivery-enhancing transporter is also a peptide, synthesis canbe achieved either using an automated peptide synthesizer or byrecombinant methods in which a polynucleotide encoding a fusion peptideis produced, as mentioned above.

EXAMPLES

The following examples are offered to illustrate, but not to limit thepresent invention.

Example 1 Penetration of Biotinylated Polymers of D-Arginine into theSkin of Nude Mice

This Example demonstrates that poly-arginine heptamers can deliverconjugated biotin into and across layers of the skin, both follicularlyand interfollicularly, and into the dermis.

Methods

Biotinylated peptides were synthesized using solid phase techniques andcommercially available Fmoc amino acids, resins, and reagents (PEBiosystems, Foster City Calif., and Bachem Torrence, Calif.) on aApplied Biosystems 433 peptide synthesizer. Fastmoc cycles were usedwith O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexyluorophosphate (HATU) substituted for HBTU/HOBt as the couplingreagent. Prior to the addition of biotin to the amino terminus of thepeptide, amino caproic acid (aca) was conjugated and acted as a spacer.The peptides were cleaved from the resin using 96% trifluoroacetic acid,2% triisopropyl silane, and 2% phenol for between 1 and 12 hours. Thelonger reaction times were necessary to completely remove the Pbfprotecting groups from the polymers of arginine. The peptidessubsequently were filtered from the resin, precipitated using diethylether, purified using HPLC reverse phase columns (Alltech Altima,Chicago, Ill.) and characterized using either electrospray or matrixassisted laser desorption mass spectrometry (Perceptive Biosystems,Boston, Mass.).

Varying concentrations (1 mM-10 μM) of a heptamer of D-arginine withbiotin covalently attached to the amino terminus using an amino caproicacid spacer (bio r7), dissolved in phosphate buffered saline (PBS), wereapplied to the back of anesthetized nude mice. Samples (100 μl) wereapplied as a liquid without excipient, prevented from dispersing by aVaseline™ barrier, and allowed to penetrate for fifteen minutes. At theend of this period the animal was sacrificed, the relevant sections ofskin were excised, embedded in mounting medium (OCT), and frozen. Frozensections (5 microns) were cut using a cryostat, collected on slides, andstained with fluorescently labeled streptavidin (Vector Laboratories,Burlingame, Calif.). The slides were fixed in acetone at 4° C. for tenminutes, air dried, soaked in PBS for five minutes, blocked with normalgoat serum for five minutes, and washed with PBS for five minutes. Thesection was stained by incubation with fluorescently labeledstreptavidin at 30 μg/ml for thirty minutes, washed with PBS,counterstained with propidium iodide (1 μg/ml) for two minutes, and thesection was mounted with Vectashield™ mounting media. Slides wereanalyzed by fluorescent microscopy. Parallel studies were done usingstreptavidin-horse radish peroxidase rather thanfluorescein-streptavidin. The biotinylated peptide was visualized bytreatment of the sections with the horseradish peroxidase substratediaminobenzadine, and visualization with light microscopy.

Results

Biotinylated arginine heptamer crossed into and across the epidermis andinto the dermis. The cytosol and nuclei of all cells in the field werefluorescent, indicating penetration into virtually every cell of thenude mouse skin in the section. The staining pattern was consistent withunanticipated transport that was both follicular and interfollicular. Inaddition, positive cells were apparent in papillary and reticulardermis. In contrast, no staining was apparent in mice treated withbiotin alone, or phosphate buffered saline alone.

Example 2 Penetration of Biotinylated Polymers of D-Arginine into theSkin of Normal Balb/C Mice

Varying concentrations (1 mM-100 μM) of a heptamer of D-arginine withbiotin covalently attached to the amino terminus using an amino caproicacid spacer (bio r7), dissolved in PBS, were applied to a skin of thegroin of an anesthetized Balb/C mice. Sample (100 μl) was applied as aliquid within excipient and prevented from dispersing by a Vaseline™bather and allowed to penetrate for thirty minutes. At the end of thisperiod animal was sacrificed, the relevant section of skin was excised,embedded in mounting medium (OCT) and frozen. Frozen sections were cutusing a cryostat, collected on slides, and stained with fluorescentlylabeled streptavidin (Vector Laboratories, Burlingame, Calif.) asdescribed in Example 1. Slides were analyzed by fluorescent microscopy.

Results

As with the skin from nude mice, biotinylated arginine heptamer crossedinto and across the epidermis and into the dermis. The cytosol andnuclei of all cells in the field were fluorescent, indicatingpenetration into virtually every cell of the nude mouse skin in thesection. The staining pattern was consistent with unanticipatedtransport that was both follicular and interfollicular. In addition,positive cells were apparent in papillary and reticular dermis. Incontrast, no staining was apparent in mice treated with biotin alone, orphosphate buffered saline alone.

Example 3 Penetration of Biotinylated Polymers of D-Arginine into NormalHuman Skin Grafted onto Nude Mice

Varying concentrations (1 mM-100 μM) of a heptamer of D-arginine withbiotin covalently attached to the amino terminus using an amino caproicacid spacer (bio r7), dissolved in PBS, were applied to human foreskingrafts on the back of SCID mice (see, e.g., Deng et al. (1997) NatureBiotechnol. 15: 1388-1391; Khavari et al. (1997) Adv. Clin. Res.15:27-35; Choate and Khavari (1997) Human Gene Therapy 8:895-901).Samples (100 μl) were applied as a liquid within excipient and preventedfrom dispersing by a Vaseline™ barrier and allowed to penetrate forfifteen minutes. At the end of this period animal was sacrificed, therelevant section of skin was excised, embedded in mounting medium (OCT)and frozen. Frozen sections were cut using a cryostat, collected onslides, and stained with fluorescently labeled streptavidin (VectorLaboratories, Burlingame, Calif.) as described in Example 1. Slides wereanalyzed by fluorescent microscopy.

Results

As with the skin from nude and normal mice, biotinylated arginineheptamer crossed into and across the epidermis and into the dermis ofthe human skin. The cytosol and nuclei of all cells in the field werefluorescent, indicating penetration into and through the epidermis anddermis. Intense staining was seen at both 20× and 40× magnification. Thestaining pattern was consistent with unanticipated transport that wasboth follicular and interfollicular. In addition, positive cells wereapparent in papillary and reticular dermis. In contrast, no staining wasapparent in mice treated with biotin alone, or phosphate buffered salinealone, and very little staining was observed with the biotinylatedarginine pentamer conjugate, either at low or high magnification.

Example 4 Increased Penetration of Biotinylated Polymers of D-Arginineinto Skin of Nude Mouse Using Plastic Wrap or a Lotion Excipient

Varying concentrations (1 mM-100 μM) of a heptamer of D-arginine withbiotin covalently attached to the amino terminus using an amino caproicacid spacer (bio r7), dissolved in PBS, and mixed with an equal volumeof Lubriderm™. The lotion mixture was then applied to the back of nudemice and allowed to penetrate for thirty, sixty, and 120 minutes.Alternatively, sample (100 μl) was applied as a liquid without excipientand prevented from evaporating by wrapping plastic wrap over the samplesealed with Vaseline™. The samples were allowed to penetrate for thirty,sixty, and 120 minutes. At the end of this period animal was sacrificed,the relevant section of skin was excised, embedded in mounting medium(OCT) and frozen. Frozen sections were cut using a cryostat, collectedon slides, and stained with fluorescently labeled streptavidin (VectorLaboratories, Burlingame, Calif.) as described in Example 1. Slides wereanalyzed by fluorescent microscopy.

Results

Both lotion and plastic wrap resulted in increased uptake compared withstaining without excipient. Lotion was more effective than plastic wrapin enhancing uptake of the conjugate. Biotinylated arginine pentamerscrossed into and across several skin layers, reaching both the cytosoland nuclei of epidermal cell layers, both follicular andinterfollicular. In addition, positive cells were apparent in papillaryand reticular dermis.

Example 5 Penetration of Cyclosporin Conjugated to a BiotinylatedPentamer, Heptamer, and Nonamer of D-Arginine into the Skin of Nude Mice

Methods

A. Linking Cyclosporin to Delivery-Enhancing Transporters

1. Preparation of the α-Chloroacetyl Cyclosporin A Derivative.

The α-chloroacetyl cyclosporin A derivative was prepared as shown inFIG. 1. Cyclosporin A (152.7 mg, 127 μmol) and chloroacetic acidanhydride (221.7 mg; 1300 μmol) were placed into a dry flask underN₂-atmosphere. Pyridine (1.0 mL) was added and the solution was heatedto 50° C. (oil bath). After 16 hours the reaction was cooled to roomtemperature and quenched with water (4.0 mL). The resulting suspensionwas extracted with diethylether (Σ 15 mL). The combined organic layerswere dried over MgSO₄. Filtration and evaporation of solvents in vacuodelivered a yellow oil, which was purified by flash chromatography onsilica gel (eluent: EtOAc/hexanes: 40%-80%) yielding 136 mg (106.4 μmol,83%) of the desired product.

2. Coupling to Transporter Molecules

A general procedure for the coupling of cysteine containing peptides tothe α-chloro acetyl Cysclosporin A derivative is shown in FIG. 2.

a. Labeled Peptides

The cyclosporin A derivative and the labeled peptide (1 equivalent) weredissolved in DMF (˜10 mmol of Cyclosporin A derivative/in L DMF) underan N₂-atmosphere. Diisopropylethylamine (10 equivalents) was added andstirring at room temperature was continued until all starting materialwas consumed (usually after 16 hours) (FIG. 3). The solvents wereremoved in vacuo and the crude reaction product was dissolved in waterand purified by reversed phase high pressure liquid chromatography(RP-HPLC) (eluent:water/MeCN*TFA). The products were obtained in thefollowing yields:

B-aca-r5-Ala-Ala-Cys-O-acyl-Cyclosporin A: 47%

B-aca-r7-Cys-O-acyl-Cyclosporin A: 43%

B-aca-r9-Cys-O-acyl-Cyclosporin A: 34%

B-aca-Cys-O-acyl-Cyclosporin A: 55%

b. Unlabeled Peptides

The peptide (34.7 mg, 15.3 μmol) and the Cyclosporin A derivative (19.6mg, 15.3 μmol) were dissolved in DMF (1.0 mL) under an N₂-atmosphere(FIG. 4). Diisopropylethylamine (19.7 mg, 153 μmol) was added andstirring at room temperature was continued. After 12 hours the solventwas removed in vacuo. The crude material was dissolved in water andpurified by RP-HPLC (eluent: water/MeCN*TFA) yielding the pure product(24.1 mg, 6.8 mmol, 44%).

B. Analysis of Transport Across Skin

Varying concentrations (1 mM-100 μM) of cyclosporin conjugated to eitherbiotinylated pentamer, heptamer, or nonamers of D-arginine (bio r5, r7,or r9), dissolved in PBS, were applied to the back of nude mice. Samples(100 μl) were applied as a liquid within excipient and prevented fromdispersing by a Vaseline™ barrier and allowed to penetrate for thirty,sixty, and 120 minutes. At the end of this period animal was sacrificed,the relevant section of skin was excised, embedded in mounting medium(OCT) and frozen. Frozen sections were cut using a cryostat, collectedon slides, and stained with fluorescently labeled streptavidin (VectorLaboratories, Burlingame, Calif.) as described in Example 1. Slides wereanalyzed by fluorescent microscopy

Results

The conjugates of cyclosporin with biotinylated heptamers and nonamersof D-arginine effectively entered into and across the epidermis and intothe dermis of the skin of nude mice. In contrast, very little uptake wasseen using a conjugate between a pentamer of arginine and cyclosporin,and no staining was seen with a PBS control. The cytosol and nuclei ofall cells in the field were fluorescent, indicating penetration into andthrough the epidermis and dermis. The staining pattern was consistentwith unanticipated transport that was both follicular andinterfollicular. In addition, positive cells were apparent in papillaryand reticular dermis. These results demonstrate remarkable uptake onlywhen sufficient guanidinyl groups are included in the delivery-enhancingtransporter.

Example 6 Demonstration that a D-Arginine Heptamer can Penetrate HumanSkin

Human and murine skin differ significantly in a number of ways, withhuman epidermis being considerably thicker. To determine if theD-arginine heptamers/cyclosporin A (r7 CsA) conjugate could alsopenetrate human skin, biotin r7 CsA was applied to full thickness humanskin grafted onto the back of a SCID mouse. As in murine skin,conjugated cyclosporin A penetrated human epidermis and dermis.Fluorescence was observed in both the cytosol and the nuclei of cells intissue exposed to biotinylated peptides alone, but in sections stainedwith biotin r7 CsA the majority of fluorescence was cytosolic,consistent with r7 CsA binding to cyclosporin A′ s known cytoplasmictargets.

Example 7 Demonstration that Cyclosporin A-Transporter Conjugates EnterT Cells in the Dermis

Methods

Inhibition of IL-2 Secretion by Releasable R7-CsA Conjugate.

Jurkat cells (5×10⁴) were incubated with varying concentrations of anonreleasable or releasable R7-CsA conjugate or CsA overnight at 37° C.to allow for the release of the active form of CsA prior to stimulationwith PMA and ionomycin. T cells subsequently were stimulated to produceIL-2 by addition of 10 ng/ml PMA (Sigma, St. Louis, Mo.) and 1 μMionomycin (CalBiochem, San Diego, Calif.). Cultures were incubatedovernight at 37° C. and supernatants were collected and IL-2 wasMeasured using a fluorescent ELISA. Briefly, plates were coated with 4μg/ml anti-human IL-2 antibody (BD Pharmingen, San Diego, Calif.),blocked with PBS containing 10% FBS for 1 hour at room temperature,washed, and supernatants added and incubated for 1 hour. Media wasremoved and biotinylated anti-human IL-2 (1.6 μg/ml), was added for onehour. The plates were washed, and then europium labeled streptavidin(0.04 ng/ml) was added for one hour. After another wash, enhancementsolution was added and the resulting fluorescence was measured using aWallac plate reader (Wallac, Turku, Finland).

Results

To determine whether biotinylated D-arginine heptamer-cyclosporin (r7CsA) conjugate would reach infiltrating T cells within inflamed skin invivo, biotin r7 CsA was applied to the site of inflammation on the backof a mouse with experimentally induced contact dermatitis. Inflamed skinwas stained with rhodamine labeled goat anti-mouse CD3 to localizeT-cells and with fluorescein labeled streptavidin to localize the biotinr7 CsA. Biotin r7 CsA was found in all CD3[+] T cells in the tissue inaddition to a variety of other cells that probably represent otherinflammatory cells as well as resident fibroblasts. These data indicatethat biotin r7 CsA penetrates inflamed skin to reach key target Tlymphocytes.

Example 8 Synthesis, In Vitro and In Vivo Activity of a ReleasableConjugate of a Short Oligomer of Arginine and CsA

Modification of the 2° alcohol of Cyclosporin A results in significantloss of its biological activity. See, e.g., R. E. Handschumacher et al,Science 226, 544-7 (1984). Consequently, to ensure release of freeCyclosporin A from its conjugate after transport into cells, CyclosporinA was conjugated to an oligo-arginine transporter through a pH sensitivelinker as shown in FIG. 10. The resultant conjugate is stable at acidicpH but at pH>7 it undergoes an intramolecular cyclization involvingaddition of the free amine to the carbonyl adjacent to Cyclosporin A(FIG. 6), which results in the release of unmodified Cyclosporin A.

Another modification in the design of the releasable conjugate was theuse of L-arginine (R), and not D-arginine (r) in the transporter. Whilethe oligo-D-arginine transporters were used for the histologicalexperiments to ensure maximal stability of the conjugate and thereforeaccuracy in determining its location through fluorescence, oligomers ofL-arginine were incorporated into the design of the releasable conjugateto minimize its biological half-life. Consistent with its design, theresultant releasable conjugate was shown to be stable at acidic pH, butlabile at physiological pH in the absence of serum. This releasableCyclosporin A conjugate's half-life in pH 7.4 PBS was 90 minutes.

Results

The releasable guanidino-heptamer conjugate of Cyclosporin A was shownto be biologically active by inhibiting IL-2 secretion by the human Tcell line, Jurkat, stimulated with PMA and ionomycin in vitro. See R.Wiskocil, et al., J Immunol 134, 1599-603 (1985). The conjugate wasadded 12 hours prior to the addition of PMA/ionomycin and dose dependentinhibition was observed by the releasable R7 CsA conjugate. Thisinhibition was not observed with a nonreleasable analog (FIG. 6) thatdiffered from the releasable conjugate by retention of the t-Bocprotecting group, which prevented cyclization and resultant release ofthe active drug. The EC₅₀ of the releasable R7 cyclosporin conjugate wasapproximately two fold higher than CsA dissolved in alcohol and added atthe same time as the releasable conjugate.

The releasable R7CsA conjugate was assayed in vivo for functionalactivity using a murine model of contact dermatitis. Treatment with the1% releasable R7 CSA conjugate resulted in 73.9%±4.0 reduction in earinflammation (FIG. 7). No reduction in inflammation was seen in theuntreated ear, indicating that the effect seen in the treated ear waslocal and not systemic. Less inhibition was observed in the ears of micetreated with 0.1 and 0.01% R7-CsA (64.8%±4.0 and 40.9%±3.3respectively), demonstrating that the effect was titratable. Treatmentwith the fluorinated corticosteroid positive control resulted inreduction in ear swelling (34.1%±6.3), but significantly less than thatobserved for 0.1% releasable R7 CsA (FIG. 7). No reduction ofinflammation was observed in any of the mice treated with unmodifiedCyclosporin A, vehicle alone, R7, or nonreleasable R7 CsA.

Example 9 The Penetration of Copper and Gadolinium-DTPA-r7 Complexesinto the Skin of Nude Mice Methods 1. Preparation of Metal ComplexesStep 1—Preparation of Copper-Diethylenetriaminepentaacetic Acid Complex(Cu-DTPA)

Copper carbonate (10 mmol) and diethylenetriaminpentacetic acid (10mmol) were dissolved in water (150 mL) (FIG. 8). After 18 h, thesolution was centrifuged to removed any solids. The blue solution wasdecanted and lyophilized to provide a blue powder (yields >90%).

Step 2—Preparation of DTPA transporter

The Cu-DTPA was linked to a transporter through an aminocaproic acidspacer using a PE Applied Biosystems Peptide Synthesizer (ABI 433A)(FIG. 9). The material was cleaved from the resin by treatment withtrifluoroacetic acid (TFA) (40 mL), triisopropyl silane (100 μL) andphenol (100 μL) for 18 h. The resin was filtered off and the peptide wasprecipitated by addition of diethyl ether (80 mL). The solution wascentrifuged and the solvent decanted off. The crude solid was purifiedby reverse-phase HPLC using a water/acetonitrile gradient. Treatmentwith TFA resulted in loss of Cu²⁺ ion which needed to be reinserted.

DTPA-aca-R7-CO2H (10 mg, 0.0063 mmol) and copper sulfate (1.6 mg, 0.0063mmol) were dissolved in water (1 mL). Let gently stir for 18 h andlyophilized to provide product as a white powder (10 mg).

2. Analysis of Transport Across Skin

Metal diethylenetriaminepentaacetic acid (DTPA) complexes were formed bymixing equimolar amounts of metal salts with DTPA in water for 18 hours.At the end of this time, the solutions were centrifuged, frozen andlyophilized. The dried powder was characterized by mass spectrometry andused in solid phase peptide synthesis. The metal-DTPA complexes wereattached to polymers of D- or L-arginine that were still attached tosolid-phase resin used in peptide synthesis. The metal-DTPA complexeswere attached using an aminocaproic acid spacer. The solid phase peptidesynthesis techniques were described in Example 1, with the exceptionthat cleavage of the peptide-DTPA-metal complex in trifluoroacetic acidreleased the metal. The metal is replaced after HPLC purification andlyophilization of the peptide-DTPA complex. Replacement of the metalinvolved incubation of equimolar amounts of the metal salt with thepeptide-aminocaproic acid-DTPA complex and subsequent lyophilization.

Varying concentrations (1 μM to 1 mM) of the Cu-DTPA-aca-r7 complex wereapplied to the abdominal region of nude mice for 15, 30 and 45 minutes.As controls, an equimolar amount of the Cu-DTPA complex was spotted ontothe abdominal region. At the end of the incubation period, the sampleswere simply wiped off and intense blue color was apparent on the skinwhere the Cu-DTPA-aca-r7 complex was spotted and not where the Cu-DTPAalone was spotted. In the case of the application of 1 mM, visible bluedye was seen for three days, decreasing with time, but being apparentfor the full period.

Varying concentrations (1 μM to 1 mM) of the Gd-DTPA-aca-r7 complex areinjected into the tail vein of BALB/c mice in 100 μl. Distribution ofthe Gd is observed in real time using magnetic resonance imaging.Distribution of the dye is apparent throughout the bloodstream, enteringliver, spleen, kidney, and heart. When injected into the carotid arteryof rabbits, the dye is seen to cross the blood brain barrier.

Example 10 Penetration of Hydrocortisone Conjugated to a BiotinylatedPentamer, Heptamer, and Nonamer of D-Arginine into the Skin of Nude MiceMethods A. Linking of Hydrocortisone to Delivery-Enhancing TransportersStep 1—Acylation of Hydrocortisone with Chloroacetic Anhydride

A solution of hydrocortisone (200 mg, 0.55 mmol) and chloroaceticanhydride (113 mg, 0.66 mmol) in pyridine (5 mL) was stirred at roomtemperature for 2 h (FIG. 10). The solvent was evaporated off and thecrude product was chromatographed on silica using 50% hexanes/ethylacetate as the eluent. Product isolated a whites solid (139 mg, 58%).

Step 2—Linking to Transporter

A solution of the chloroacetic ester of hydrocortisone (0.0137 mmol), atransporter containing a cysteine residue (0.0137) anddiisopropylethylamine (DIEA) (0.0274 mmol) in dimethylformamide (DMF) (1mL) was stirred at room temperature for 18 h (FIG. 11). The material waspurified via reverse-phase HPLC using a water/acetonitrile gradient andlyophilized to provide a white powder.

r5 conjugate-12 mg obtained (29% isolated yield)

r7 conjugate-22 mg obtained (55% isolated yield)

R7 conjugate-13 mg obtained (33% isolated yield)

B. Analysis of Transport Across Skin

Varying concentrations (1 mM-100 μM) of hydrocortisone conjugated toeither biotinylated pentamer, heptamer, or nonamers of D-arginine (bior5, r7, or r9), dissolved in PBS, were applied to the back of nude mice.Samples (100 μl) were applied as a liquid within excipient and preventedfrom dispersing by a Vaseline™ barrier and allowed to penetrate forthirty, sixty, and 120 minutes. At the end of this period animal wassacrificed, the relevant section of skin was excised, embedded inmounting medium (OCT) and frozen. Frozen sections were cut using acryostat, collected on slides, and stained with fluorescently labeledstreptavidin (Vector Laboratories, Burlingame, Calif.) as described inExample 1. Slides were analyzed by fluorescent microscopy.

Results

The conjugates of hydrocortisone with biotinylated heptamers ofD-arginine effectively entered into and across the epidermis and intothe dermis of the skin of nude mice. In contrast, very little uptake wasseen using a conjugate between a pentamer of arginine andhydrocortisone, and no staining was seen with a PBS control. The cytosoland nuclei of all cells in the field were fluorescent, indicatingpenetration into and through the epidermis and dermis. The stainingpattern was consistent with unanticipated transport that was bothfollicular and interfollicular. In addition, positive cells wereapparent in papillary and reticular dermis. These results demonstrateremarkable uptake only when sufficient guanidinyl groups are included inthe delivery-enhancing transporter.

Example 11

Penetration of Taxol Conjugated to a Biotinylated Pentamer, Heptamer,and Nonamer of D-Arginine into the Skin of Nude Mice

Methods 1. Conjugation of C-2′ Activated Taxol Derivatives toBiotin-Labeled Peptides Synthesis of C-2′ Derivatives

Taxol (48.7 mg, 57.1 μmol) was dissolved in CH₂Cl₂ (3.0 mL) under anN₂-atmosphere. The solution was cooled to 0° C. A stock solution of thechloroformate of benzyl-(p-hydroxy benzoate) (200 mmol, in 2.0 mLCH₂Cl₂— freshly prepared from benzyl-(p-hydroxy benzoate) anddiphosgene) was added at 0° C. and stirring at that temperature wascontinued for 5 hours, after which the solution was warmed to roomtemperature (FIG. 12). Stirring was continued for additional 10 hours.The solvents were removed in vacuo and the crude material was purifiedby flash chromatography on silica gel (eluent: EtOAc/hexanes 30%-70%)yielding the desired taxol C-2′ carbonate (36.3 mg, 32.8 μmol, 57.4%).

Coupling to Biotin-Labeled Peptides.

A procedure for coupling to biotin-labeled peptides is shown in FIG. 13.The taxol derivative and the biotin labeled peptide (1.2 equivalents)were dissolved in DMF (˜10 μmol/mL DMF) under an N₂-atmosphere. Stocksolutions of diisopropylethylamine (1.2 equivalents in DMF) and DMAP(0.3 equivalents in DMF) were added and stirring at room temperature wascontinued until all starting material was consumed. After 16 hours thesolvent was removed in vacuo. The crude reaction mixture was dissolvedin water and purified by RP-HPLC (eluent: water/MeCN*TFA) yielding theconjugates in the indicated yields:

B-aca-r5-K-taxol: 3.6 mg, 1.32 mmol, 20%.

B-aca-r7-K-taxol: 9.8 mg, 3.01 mmol, 44%.

B-aca-r9-K-taxol: 19.4 mg, 5.1 mmol, 67%.

Unlabeled C-2′ Carbamates:

The taxol derivative (12.4 mg, 11.2 μmol) and the unlabeled peptide(27.1 mg, 13.4 μmol) were dissolved in DMF (1.5 mL) under anN₂-atmosphere (FIG. 14). Diisopropylethylamine (1.7 mg, 13.4 μmol) wasadded as a stock solution in DMF, followed by DMAP (0.68 mg, 5.6 μmol)as a stock solution in DMF. Stirring at room temperature was continueduntil all starting material was consumed. After 16 hours the solvent wasremoved in vacuo. The crude material was dissolved in water and purifiedby RP-HPLC (eluent: water/MeCN*TFA) yielding the desired product (16.5mg, 5.9 μmol, 53%).

Other C-2′ Conjugates

The taxol derivative (8.7 mg, 7.85 mop was dissolved in EtOAc (2.0 mL).Pd/C (10%, 4.0 mg) was added and the reaction flask was purged with H₂five times (FIG. 15A). Stirring under an atmosphere of hydrogen wascontinued for 7 hours. The Pd/C was filtered and the solvent was removedin vacuo. The crude material (6.7 mg, 6.58 μmol, 84%) obtained in thisway was pure and was used in the next step without further purification.

The free acid taxol derivative (18.0 mg, 17.7 μmol) was dissolved inCH₂Cl₂ (2.0 mL). Dicyclohexylcarbodiimide (4.3 mg, 21.3 μmol) was addedas a stock solution in CH₂Cl₂ (0.1 mL). N-Hydroxysuccinimide (2.0 mg,17.7 μmol) was added as a stock solution in DMF (0.1 mL) (FIG. 15B).Stirring at room temperature was continued for 14 hours. The solvent wasremoved in vacuo and the resultant crude material was purified by flashchromatography on silica gel (eluent: EtOAc/hexanes 40%-80%) yieldingthe desired product (13.6 mg, 12.2 μmol, 69%).

The activated taxol derivative (14.0 mg, 12.6 μmol) and the peptide(30.6 mg, 15.1 μmol) were dissolved in DMF (3.0 mL) under anN₂-atmosphere (FIG. 15C). Diisopropylethylamine (1.94 mg, 15.1 μmol) wasadded as a stock solution in DMF (0.1 mL), followed by DMAP (0.76 mg,6.3 μmol) as a stock solution in DMF 0.1 mL). Stirring at roomtemperature was continued until all the starting material was consumed.After 20 hours the solvent was removed in vacuo. The crude material wasdissolved in water and purified by RP-HPLC (eluent: water/MeCN*TFA)yielding the two depicted taxol conjugates in a ration of 1:6 (carbonatevs carbamate, respectively).

2. Analysis of Transport Across Skin

Varying concentrations (1 mM-100 μM) of taxol conjugated to eitherbiotinylated pentamer, heptamer, or nonamers of D-arginine (bio r5, r7,or r9), dissolved in PBS, were applied to the back of nude mice. Samples(100 μl) were applied as a liquid within excipient and prevented fromdispersing by a Vaseline™ barrier and allowed to penetrate for thirty,sixty, and 120 minutes. At the end of this period animal was sacrificed,the relevant section of skin was excised, embedded in mounting medium(OCT) and frozen. Frozen sections were cut using a cryostat, collectedon slides, and stained with fluorescently labeled streptavidin (VectorLaboratories, Burlingame, Calif.) as described in Example 1. Slides wereanalyzed by fluorescent microscopy.

Results

The conjugates of taxol with biotinylated heptamers and nonamers ofD-arginine effectively entered into and across the epidermis and intothe dermis of the skin of nude mice. In contrast, very little uptake wasseen using a conjugate between a pentamer of arginine and taxol, and nostaining was seen with a PBS control. The cytosol and nuclei of allcells in the field were fluorescent, indicating penetration into andthrough the epidermis and dermis. The staining pattern was consistentwith unanticipated transport that was both follicular andinterfollicular. In addition, positive cells were apparent in papillaryand reticular dermis. These results demonstrate remarkable uptake onlywhen sufficient guanidinyl groups are included in the delivery-enhancingtransporter.

Example 12

Conjugate of Taxol and Delivery-Enhancing Transporter with pH-ReleasableLinker

This Example demonstrates the use of a general strategy for synthesizingprodrugs that have a delivery-enhancing transporter linked to a drug bya linker that releases the drug from the delivery-enhancing transporterupon exposure to physiological pH. In general, a suitable site on thedrug is derivatized to carry an α-chloroacetyl residue. Next, thechlorine is displaced with the thiol of a cysteine residue that carriesan unprotected amine. This scheme is shown in FIG. 16.

Methods Synthesis of Taxol-2′-chloroacetyl

Taxol (89.5 mg, 104.9 μmol) was dissolved in CH₂Cl₂ (3.5 mL). Thesolution was cooled to 0° C. under an N₂-atmosphere. α-Chloroaceticanhydride (19.7 mg, 115.4 μmol) was added, followed by DMA (14.8 mg,115.4 μmol). The solution was allowed to warm to room temperature. Afterthin layer chromatography (tlc) analysis indicated complete consumptionof starting material, the solvent was removed in vacuo and the crudematerial was purified by flash chromatography on silica gel (eluent:EtOAC/Hex 20%-50%) yielding the desired material (99.8 mg, quantitative)(FIG. 18).

¹H-NMR (CDCl₃): δ=8.13 (d, J=7.57 Hz, 2H), 7.72 (d, J=7.57 Hz, 2H),7.62-7.40 (m, 11H), 6.93 (d, J=9.14 Hz, 1H), 6.29-6.23 (m, 2H), 6.01 (d,J=7.14 Hz, 1H), 5.66 (d, J=6.80 Hz, 1H), 5.55 (d, J=2.24 Hz, 1H), 4.96(d, J=8.79 Hz, 1H), 4.43 (m, 1H), 4.30 (d, J=8.29 Hz, 1H), 4.20-4.15 (m,2H), 3.81 (d, J=6.71 Hz, 1H), 2.56-2.34 (m, 3H), 2.45 (s, 3H), 2.21 (s,3H), 2.19 (m, 1H), 1.95-1.82 (m, 3H), 1.92 s, (3H), 1.67 (s, 3H), 1.22(s, 3H), 1.13 (s, 3H) ppm.

¹³C-NMR (CDCl₃): δ=203.6, 171.1, 169.7, 167.3, 167.0, 166.9, 166.3,142.3, 136.4, 133.6, 133.5, 132.9, 132.0, 130.1, 129.2, 121.1, 128.7,128.6, 127.0, 126.5, 84.3, 81.0, 79.0, 76.3, 75.4, 75.2, 75.0, 72.2,72.0, 58.4, 52.7, 45.5, 43.1, 40.1, 35.5, 26.7, 22.6, 22.0, 20.7, 14.7,9.5 ppm.

Linkage of Taxol to Delivery-Enhancing Transporter

The peptide (47.6 mg, 22.4 μmol) was dissolved in DMF (1.0 mL) under anN₂-atmosphere. DIEA (2.8 mg, 22.4 μmol) was added. A solution oftaxol-2′-chloroacetate (20.8 mg, 22.4 μmol) in DMF (1.0 mL) was added.Stirring at room temperature was continued for 6 hours. Water containing0.1% TFA (1.0 mL) was added, the sample was frozen and the solvents werelyophilized. The crude material was purified by RP-HPLC (eluent:water/MeCN*0.1% TFA: 85%-15%). A schematic of this reaction is shown inFIG. 18.

Synthesis of Related conjugates

Using the conjugation conditions outlined above, the three additionalconjugates shown in were synthesized.

Cytotoxicity Assay

The taxol conjugates were tested for cytotoxicity in a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT) dyereduction. Results, which are shown in FIG. 20, demonstrate that thetaxol conjugated to r7 with a readily pH-releasable linker (CG 1062;R=Ac in the structure shown in FIG. 19) is significantly more cytotoxicthan either taxol alone or taxol conjugated to r7 with a less-readilypH-releasable linker (CG 1040; R═H in the structure shown in FIG. 19).

Example 13 Structure-Function Relationships of Fluorescently-LabeledPeptides Derived from Tat₄₉₋₅₇

Methods

General.

Rink amide resin and Boc₂O were purchased from Novabiochem.Diisopropylcarbodiimide, bromoacetic acid, fluorescein isothiocyanate(FITC-NCS), ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane,1,6-diaminohexane, trans-1,6-diaminocyclohexane, andpyrazole-1-carboxamidine were all purchased from Aldrich®. All solventsand other reagents were purchased from commercial sources and usedwithout further purification. The mono-Boc amines were synthesized fromthe commercially available diamines using a literature procedure (10equiv. of diamine and 1 equiv. of Boc₂O in chloroform followed by anaqueous work up to remove unreacted diamine) (34).

N-tert-butoxycarbonyl-1,6-trans-diaminocyclohexane.

Mp 159-161° C.; ¹H NMR (CDCl₃) δ 4.35 (br s, 1H), 3.37 (br s, 1H), 2.61(br s, 1H), 1.92-2.02 (m, 2H), 1.81-1.89 (m, 2H), 1.43 (s, 9H),1.07-1.24 (m, 4H) ppm; ¹³C NMR (D₆-DMSO) δ 154.9, 77.3, 49.7, 48.9,35.1, 31.4, 28.3 ppm; ES-MS (M+1) Calcd 215.17. Found 215.22.

General Procedure for Peptide Synthesis.

Tat₄₉₋₅₇ (RKKRRQRRR), truncated and alanine-substituted peptides derivedfrom Tat₄₉₋₅₇, Antennapedia₄₃₋₅₈ (RQIKIWFQNRRMKWKK), and homopolymers ofarginine (R5-R9) and d-arginine (r5-r9) were prepared with an automatedpeptide synthesizer (ABI433) using standard solid-phase Fmoc chemistry(35) with HATU as the peptide coupling reagent. The fluorescein moietywas attached via a aminohexanoic acid spacer by treating a resin-boundpeptide (1.0 mmol) with fluorescein isothiocyanate (1.0 mmol) and DIEA(5 mmol) in DMF (10 mL) for 12 h. Cleavage from the resin was achievedusing 95:5 TFA/triisopropylsilane. Removal of the solvent in vacuo gavea crude oil which was triturated with cold ether. The crude mixture thusobtained was centrifuged, the ether was removed by decantation, and theresulting orange solid was purified by reverse-phase HPLC(H₂O/CH₃CN in0.1% TFA). The products were isolated by lyophilization andcharacterized by electrospray mass spectrometry. Purity of the peptideswas >95% as determined by analytical reverse-phase HPLC (H₂O/CH₃CN in0.1% TFA).

All peptides and peptoids synthesized contain an aminohexanoic (ahx)acid moiety attached to the N-terminal amino group with a fluoresceinmoiety (Fl) covalently linked to the amino group of the aminohexanoicacid spacer. The carboxyl terminus of every peptide and peptoid is acarboxamide.

Cellular Uptake Assay.

The arginine homopolymers and guanidine-substituted peptoids were eachdissolved in PBS buffer (pH 7.2) and their concentration was determinedby absorption of fluorescein at 490 nm (ε=67,000). The accuracy of thismethod for determining concentration was established by weighingselected samples and dissolving them in a known amount of PBS buffer.The concentrations determined by UV spectroscopy correlated with theamounts weighed out manually. Jurkat cells (human T cell line), murine Bcells (CH27), or human PBL cells were grown in 10% fetal calf serum andDMEM and each of these were used for cellular uptake experiments.Varying amounts of arginine and oligomers of guanidine-substitutedpeptoids were added to approximately 3×10⁶ cells in 2% FCS/PBS (combinedtotal of 200 μL) and placed into microtiter plates (96 well) andincubated for varying amount of time at 23° C. or 4° C. The microtiterplates were centrifuged and the cells were isolated, washed with coldPBS (3×250 μL), incubated with 0.05% trypsin/0.53 mM EDTA at 37° C. for5 min, washed with cold PBS, and resuspended in PBS containing 0.1%propidium iodide. The cells were analyzed using fluorescent flowcytometry (FACScan, Becton Dickinson) and cells staining with propidiumiodide were excluded from the analysis. The data presented is the meanfluorescent signal for the 5000 cells collected.

Inhibition of Cellular Uptake with Sodium Azide.

The assays were performed as previously described with the exceptionthat the cells used were preincubated for 30 min with 0.5% sodium azidein 2% FCS/PBS buffer prior to the addition of fluorescent peptides andthe cells were washed with 0.5% sodium azide in PBS buffer. All of thecellular uptake assays were run in parallel in the presence and absenceof sodium azide.

Cellular Uptake Kinetics Assay.

The assays were performed as previously described except the cells wereincubated for 0.5, 1, 2, and 4 min at 4° C. in triplicate in 2% FCS/PBS(50 μl) in microtiter plates (96 well). The reactions were quenched bydiluting the samples into 2% FCS/PBS (5 mL). The assays were then workedup and analyzed by fluorescent flow cytometry as previously described.

Results

To determine the structural requirements for the cellular uptake ofshort arginine-rich peptides, a series of fluorescently-labeledtruncated analogues of Tat₄₉₋₅₇ were synthesized using standardsolid-phase chemistry. See, e.g., Atherton, E. et al. SOLID-PHASEPEPTIDE SYNTHESIS (IRL: Oxford, Engl. 1989). A fluorescein moiety wasattached via an aminohexanoic acid spacer on the amino termini. Theability of these fluorescently labeled peptides to enter Jurkat cellswas then analyzed using fluorescent activated cell sorting (FACS). Thepeptide constructs tested were Tat₄₉₋₅₇ (Fl-ahx-RKKRRQRRR): Tat₄₉₋₅₆(Fl-ahx-RKKRRQRR), Tat₄₉₋₅₅ (Fl-ahx-RKKRRQR), Tat₅₀₋₅₇(Fl-ahx-KKRRQRRR), and Tat₅₁₋₅₇ (Fl-ahx-KRRQRRR). Differentiationbetween cell surface binding and internalization was accomplishedthroughout by running a parallel set of assays in the presence andabsence of sodium azide. Because sodium azide inhibits energy-dependentcellular uptake but not cell surface binding, the difference influorescence between the two assays provided she amount of fluorescenceresulting from internalization.

Deletion of one arginine residue from either the amine terminus(Tat₅₀₋₅₇) or the carboxyl terminus (Tat₄₉₋₅₆) resulted in an 80% lossof intracellular fluorescence compared to the parent sequence(Tat₄₉₋₅₇). From the one amino acid truncated analogs, further deletionof R-56 from the carboxyl terminus (Tat₄₉₋₅₅) resulted in an additional60% loss of intracellular fluorescence, while deletion of K-50 from theamine terminus (Tat₅₁₋₅₇) did not further diminish the amount ofinternalization. These results indicate that truncated analogs ofTat₄₉₋₅₇ are significantly less effective at the transcellular deliveryof fluorescein into Jurkat cells, and that the arginine residues appearto contribute more to cellular uptake than the lysine residues.

To determine the contribution of individual amino acid residues tocellular uptake, analogs containing alanine substitutions at each siteof Tat₄₉₋₅₇ were synthesized and assayed by FACS analysis (FIG. 22). Thefollowing constructs were tested: A-49 (Fl-ahx-AKKRRQRRR), A-50(Fl-ahx-RAKRRQRRR), A-51 (Fl-ahx-RKARRQRRR), A-52 (Fl-ahx-RKKARQRRR),A-53 (Fl-ahx-RKKRAQRRR), A-54 (Fl-ahx-RKKRRARRR), A-55(Fl-ahx-RKKRRQARR), A-56 (Fl-ahx-RKKRRQRAR), and A-57(Fl-ahx-RKKRRQRRA). Substitution of the non-charged glutamine residue ofTat₄₉₋₅₇ with alanine (A-54) resulted in a modest decrease in cellularinternalization. On the other hand, alanine substitution of each of thecationic residues individually produced a 70-90% loss of cellularuptake. In these cases, the replacement of lysine (A-50, A-51) orarginine (A-49, A-52, A-55, A-56, A-57) with alanine had similar effectsin reducing uptake.

To determine whether the chirality of the transporter peptide wasimportant, the corresponding d-(d-Tat₄₉₋₅₇), retro-l-(Tat₅₇₋₄₉), andretro-inverso isomers (d-Tat₅₇₋₄₉) were synthesized and assayed by FACSanalysis (FIG. 23). Importantly, all three analogs were more effectiveat entering Jurkat cells then Tat₄₉₋₅₇. These results indicated that thechirality of the peptide backbone is not crucial for cellular uptake.Interestingly, the retro-l isomer (Tat₅₇₋₄₉) which has three arginineresidues located at the amine terminus instead of one arginine and twolysines found in Tat₄₉₋₅₇ demonstrated enhanced cellular uptake. Thus,residues at the amine terminus appear to be important and that argininesare more effective than lysines for internalization. The improvedcellular uptake of the unnatural d-peptides is most-likely due to theirincreased stability to proteolysis in 2% FCS (fetal calf serum) used inthe assays. When serum was excluded, the d- and l-peptides wereequivalent as expected.

These initial results indicated that arginine content is primarilyresponsible for the cellular uptake of Tat₄₉₋₅₇. Furthermore, theseresults were consistent with our previous results where we demonstratedthat short oligomers of arginine were more effective at entering cellsthen the corresponding short oligomers of lysine, ornithine, andhistidine. What had not been established was whether argininehomo-oligomers are more effective than Tat₄₉₋₅₇. To address this point,Tat₄₉₋₅₇ was compared to the l-arginine (R5-R9) and d-arginine (r5-r9)oligomers. Although Tat₄₉₋₅₇ contains eight cationic residues, itscellular internalization was between that of R6 and R7 (FIG. 24)demonstrating that the presence of six arginine residues is the mostimportant factor for cellular uptake. Significantly, conjugatescontaining 7-9 arginine residues exhibited better uptake than Tat₄₉₋₅₇.

To quantitatively compare the ability of these arginine oligomers andTat₄₉₋₅₇ to enter cells, Michaelis-Menton kinetic analyses wereperformed. The rates of cellular uptake were determined after incubation(3° C.) of the peptides in Jurkat cells for 30, 60, 120, and 240 seconds(Table 1). The resultant K_(m) values revealed that r9 and R9 enteredcells at rates approximately 100-fold and 20-fold faster than Tat₄₇₋₅₉respectively. For comparison, Antennapedia₄₃₋₅₈ was also analyzed andwas shown to enter cells approximately 2-fold faster than Tat₄₇₋₅₉, butsignificantly slower than r9 or R9.

TABLE 1 Michaelis-Menton kinetics: Antennapedia₄₃₋₅₈ (Fl-ahx-RQIKIWFQNRRMKWKK). peptide K_(m)(μM) V_(max) Tat₄₉₋₅₇ 770 0.38Antennapedia₄₃₋₅₈ 427 0.41 R9 44 0.37 r9 7.6 0.38

Example 14 Design and Synthesis of Peptidomimetic Analogs of Tat₄₉₋₅₇

Methods

General Procedure for Peptoid Polyamine Synthesis. Peptoids weresynthesized manually using a flitted glass apparatus and positivenitrogen pressure for mixing the resin following the literatureprocedure developed by Zuckermann. See, e.g., Murphy, J. E. et al.,Proc. Natl. Acad. Sci. USA 95, 1517-1522 (1998); Simon, R. J. et al.,Proc. Natl. Acad. Sci. USA 89, 9367-9371 (1992); Zuckermann, R. J. etal., J. Am. Chem. Soc. 114, 10646-10647 (1992). Treatment ofFmoc-substituted Rink amide resin (0.2 mmol) with 20% piperidine/DMF (5mL) for 30 min (2×) gave the free resin-bound amine which was washedwith DMF (3×5 mL). The resin was treated with a solution of bromoaceticacid (2.0 mmol) in DMF (5 mL) for 30 min. This procedure was repeated.The resin was then washed (3×5 mL DMF) and treated with a solution ofmono-Boc diamine (8.0 mmol) in DMF (5 mL) for 12 hrs. These two stepswere repeated until an oligomer of the required length was obtained(Note: the solution of mono-Boc diamine in DMF could be recycled withoutappreciable loss of yield). The resin was then treated withN-Fmoc-aminohexanoic acid (2.0 mmol) and DIC (2.0 mmol) in DMF for 1 hand this was repeated. The Fmoc was then removed by treatment with 20%piperidine/DMF (5 mL) for 30 min. This step was repeated and the resinwas washed with DMF (3×5 mL). The free amine resin was then treated withfluorescein isothiocyanate (0.2 mmol) and DIEA (2.0 mmol) in DMF (5 mL)for 12 hrs. The resin was then washed with DMF (3×5 mL) anddichloromethane (5×5 mL). Cleavage from the resin was achieved using95:5 TFA/triisopropylsilane (8 mL). Removal of the solvent in vacuo gavea crude oil which was triturated with cold ether (20 mL). The crudemixture thus obtained was centrifuged, the ether was removed bydecantation, and the resulting orange solid was purified byreverse-phase HPLC(H₂O/CH₃CN in 0.1% TFA). The products were isolated bylyophilization and characterized by electrospray mass spectrometry andin selected cases by ¹H NMR spectroscopy.

General Procedure for Perguanidinylation of Peptoid Polyamines.

A solution of peptoid amine (0.1 mmol) dissolved in deionized water (5mL) was treated with sodium carbonate (5 equivalents per amine residue)and pyrazole-1-carboxamidine (5 equivalents per amine residue) andheated to 50° C. for 24-48 hr. The crude mixture was then acidified withTFA (0.5 mL) and directly purified by reverse-phase HPLC (H₂O/CH₃CN in0.1% TFA). The products were characterized by electrospray massspectrometry and isolated by lyophilization and further purified byreverse-phase HPLC. The purity of the guanidine-substituted peptoidswas >95% as determined by analytical reverse-phase HPLC (H₂O/CH₃CN in0.1% TFA).

Results

Utilizing the structure-function relationships that had been determinedfor the cellular uptake of Tat₄₇₋₅₉, we designed a set of polyguanidinepeptoid derivatives that preserve the 1,4 backbone spacing of sidechains of arginine oligomers, but have an oligo-glycine backbone devoidof stereogenic centers. These peptoids incorporating arginine-like sidechains on the amide nitrogen were selected because of their expectedresistance to proteolysis, and potential ease and significantly lowercost of synthesis (Simon et al., Proc. Natl. Acad. Sci. USA 89:9367-9371(1992); Zuckermann, et al., J. Am. Chem. Soc. 114:10646-10647 (1992).Furthermore, racemization, frequently encountered in peptide synthesis,is not a problem in peptoid synthesis; and the “sub-monomer” peptoidapproach allows for facile modification of side-chain spacers. Althoughthe preparation of an oligurea and peptoid-peptide hybrid (Hamy, et al,Proc. Natl. Acad. Sci. USA 94:3548-3553 (1997)) derivatives of Tat₄₉₋₅₇have been previously reported, their cellular uptake was not explicitlystudied.

The desired peptoids were prepared using the “sub-monomer” approach(Simon et al.; Zuckermann et al.) to peptoids followed by attachment ofa fluorescein moiety via an aminohexanoic acid spacer onto the aminetermini. After cleavage from the solid-phase resin, the fluorescentlylabeled polyamine peptoids thus obtained were converted in good yields(60-70%) into polyguanidine peptoids by treatment with excesspyrazole-1-carboxamidine (Bernatowicz, et al., J. Org. Chem.57:2497-2502 (1992) and sodium carbonate (as shown in FIG. 25).Previously reported syntheses of peptoids containing isolated N-Argunits have relied on the synthesis of N-Arg monomers (5-7 steps) priorto peptoid synthesis and the use of specialized and expensive guanidineprotecting groups (Pmc, Pbf) (Kruijtzer, et al., Chem. Eur. J.4:1570-1580 (1998); Heizmann, et al. Peptide Res. 7:328-332 (1994). Thecompounds reported here represent the first examples ofpolyguanidinylated peptoids prepared using a perguanidinylation step.This method provides easy access to polyguanidinylated compounds fromthe corresponding polyamines and is especially useful for the synthesisof perguanidinylated homooligomers. Furthermore, it eliminates the useof expensive protecting groups (Pbf, Prnc). An additional example of aperguanidinylation of a peptide substrate using a noveltriflyl-substituted guanylating agent has recently been reported(Feichtinger, et al., J. Org. Chem. 63:8432-8439 (1998)).

The cellular uptake of fluorescently labeled polyguanidine N-arg-5,7,9peptoids was compared to the corresponding d-arginine peptides r5,7,9(similar proteolytic properties) using Jurkat cells and FACS analysis.The amount of fluorescence measured inside the cells with N-arg-5,7,9was proportional to the number of guanidine residues:N-arg9>N-arg7>N-arg5 (FIG. 26), analogous to that found for r5,7,9.Furthermore, the N-arg-5,7,9 peptoids showed only a slightly loweramount of cellular entry compared to the corresponding peptides, r5,7,9.The results demonstrate that the hydrogen bonding along the peptidebackbone of Tat₄₉₋₅₇ or arginine oligomers is not a required structuralelement for cellular uptake and oligomeric guanidine-substitutedpeptoids can be utilized in place of arginine-rich peptides as moleculartransporters. The addition of sodium azide inhibited internalizationdemonstrating that the cellular uptake of peptoids was also energydependent.

Example 15 The Effect of Side Chain Length on Cellular Uptake

After establishing that the N-arg peptoids efficiently crossed cellularmembranes, the effect of side chain length (number of methylenes) oncellular uptake was investigated. For a given number of guanidineresidues (5,7,9), cellular uptake was proportional to side chain length.Peptoids with longer side chains exhibited more efficient cellularuptake. A nine-mer peptoid analog with a six-methylene spacer betweenthe guanidine head groups and the backbone (N-hxg9) exhibited remarkablyhigher cellular uptake than the corresponding d-arginine oligomer (r9).The relative order of uptake was N-hxg9 (6 methylene)>N-btg9 (4methylene)>r9 (3 methylene)>N-arg9 (3 methylene)>N-etg9 (2 methylene)(FIG. 27). Of note, the N-hxg peptoids showed remarkably high cellularuptake, even greater than the corresponding d-arginine oligomers. Thecellular uptake of the corresponding heptamers and pentamers also showedthe same relative trend. The longer side chains embodied in the N-hxgpeptoids improved the cellular uptake to such an extent that the amountof internalization was comparable to the corresponding d-arginineoligomer containing one more guanidine residue (FIG. 28). For example,the N-hxg7 peptoid showed comparable cellular uptake to r8.

To address whether the increase in cellular uptake was due to theincreased length of the side chains or due to their hydrophobic nature,a set of peptoids was synthesized containing cyclohexyl side chains.These are referred to as the N-chg-5,7,9 peptoids. These contain thesame number of side chain carbons as the N-hxg peptoids but possessdifferent degrees of freedom. Interestingly, the N-chg peptoid showedmuch lower cellular uptake activity than all of the previously assayedpeptoids, including the N-etg peptoids (FIG. 29). Therefore, theconformational flexibility and sterically unencumbered nature of thestraight chain alkyl spacing groups is important for efficient cellularuptake.

Discussion

The nona-peptide, Tat₄₉₋₅₇, has been previously shown to efficientlytranslocate through plasma membranes. The goal of this research was todetermine the structural basis for this effect and use this informationto develop simpler and more effective molecular transporters. Towardthis end, truncated and alanine substituted derivatives of Tat₄₉₋₅₇conjugated to a fluoroscein label was prepared. These derivativesexhibited greatly diminished cellular uptake compared to Tat₄₉₋₅₇,indicating that all of the cationic residues of Tat₄₉₋₅₇ are requiredfor efficient cellular uptake. When compared with our previous studieson short oligomers of cationic oligomers, these findings suggested thatan oligomer of arginine might be superior to Tat₄₉₋₅₇ and certainly moreeasily and cost effectively prepared. Comparison of short arginineoligomers with Tat₄₉₋₅₇ showed that members of the former were indeedmore efficiently taken into cells. This was further quantified for thefirst time bt Michaelis-Menton kinetics analysis which showed that theR9 and r9 oligomers had Km values 30-fold and 100-fold greater than thatfound for Tat₄₉₋₅₇.

Given the importance of the guanidino head group and the apparentinsensitivity of the oligomer chirality revealed in our peptide studies,we designed and synthesized a novel series of polyguanidine peptoids.The peptoids N-arg-5,7,9, incorporating the arginine side chain,exhibited comparable cellular uptake to the corresponding d-argininepeptides r5,7,9, indicating that the hydrogen bonding along the peptidebackbone and backbone chirality are not essential for cellular uptake.This observation is consistent with molecular models of these peptoids,arginine oligomers, and Tat₄₉₋₅₇, all of which have a deeply embeddedbackbone and a guanidinium dominated surface. Molecular models furtherreveal that these structural characteristics are retained in varyingdegree in oligomers with different alkyl spacers between the peptoidbackbone and guanidino head groups. Accordingly, a series of peptoidsincorporating 2-(N-etg), 4-(N-btg), and 6-atom (N-hxg) spacers betweenthe backbone and side chain were prepared and compared for cellularuptake with the N-arg peptoids (3-atom spacers) and d-arginineoligomers. The length of the side chains had a dramatic affect oncellular entry. The amount of cellular uptake was proportional to thelength of the side chain with N-hxg>N-btg>N-arg>N-etg. Cellular uptakewas improved when the number of alkyl spacer units between the guanidinehead group and the backbone was increased. Significantly, N-hxg9 wassuperior to r9, the latter being 100-fold better than Tat₄₉₋₅₇. Thisresult led us to prepare peptoid derivatives containing longer octylspacers (N-ocg) between the guanidino groups and the backbone. Issuesrelated to solubility prevented us from testing these compounds.

Because both perguanidinylated peptides and perguanidinylated peptoidsefficiently enter cells, the guanidine head group (independent ofbackbone) is apparently the critical structural determinant of cellularuptake. However, the presence of several (over six) guanidine moietieson a molecular scaffold is not sufficient for active transport intocells as the N-chg peptoids did not efficiently translocate into cells.Thus, in addition to the importance of the guanidine head group, thereare structure/conformational requirements that are significant forcellular uptake.

In summary, this investigation identified a series of structuralcharacteristics including sequence length, amino acid composition, andchirality that influence the ability of Tat₄₉₋₅₇ to enter cells. Thesecharacteristics provided the blueprint for the design of a series ofnovel peptoids, of which 17 members were synthesized and assayed forcellular uptake. Significantly, the N-hxg9 transporter was found to besuperior in cell uptake to r9 which was comparable to N-btg9. Hence,these peptoid transporters proved to be substantially better thanTat₄₉₋₅₇. This research established that the peptide backbone andhydrogen bonding along that backbone are not required for cellularuptake, that the guanidino head group is superior to other cationicsubunits, and most significantly, that an extension of the alkyl chainbetween the backbone and the head group provides superior transporters.In addition to better uptake performance, these novel peptoids offerseveral advantages over Tat₄₉₋₅₇ including cost-effectiveness, ease ofsynthesis of analogs, and protease stability. These features along withtheir significant water solubility (>100 mg/mL) indicate that thesenovel peptoids could serve as effective transporters for the moleculardelivery of drugs, drug candidates, and other agents into cells.

Example 16 Synthesis of Itraconazole-Transporter Conjugate

This Example provides one application of a general strategy forattaching a delivery-enhancing transporter to a compound that includes atriazole structure. The scheme, using attachment of itraconazole to anarginine (r7) delivery-enhancing transporter as an example, is shown inFIG. 30. In the scheme, R is H or allyl, n is 1 or 2, and X is ahalogen.

The reaction involves making use of quaternization of a nitrogen in thetriazole ring to attach an acyl group that has a halogen (e.g., Br, Fl,I) or a methyl ester. Compound 3 was isolated by HPLC. Proton NMR in D₂Orevealed itraconazole and transporter peaks.

The methyl ester provided yields of 70% and greater, while yieldsobtained using the Br-propionic acid/ester pair were 40-50%. Theacylderivative is then reacted with the amine of the delivery-enhancingtransporter to form the conjugate. Alternatively, the halogenated acylgroup can first be attached to the transporter molecule through an amidelinkage, after which the reaction with the drug compound is conducted.

Example 17 Preparation of FK506 Conjugates

This Example describes the preparation of conjugates in which FK506 isattached to a delivery-enhancing transporter. Two different linkers wereused, each of which released FK506 at physiological pH (pH 5.5 to 7.5),but had longer half-lives at more acidic pH. These schemes arediagrammed in FIGS. 31A and B.

Linker 1 6-maleimidocaproic hydrazide trifluoroacetate (Scheme I and II)

A solution of FK506 (1) (0.1 g, 124.4 μmol), 6-maleiimidocaproichydrazide trifluoroacetate (2) (0.126 g, 373.2 μmol) and trifluoroaceticacid (catalytic, 1 μl) in anhydrous methanol (5 mL) was stirred at roomtemperature for 36 h. The reaction was monitored by thin layerchromatography that showed almost complete disappearance of the startingmaterial. [TLC solvent system—dichloromethane (95):methanol (5),R_(f)=0.3]. The reaction mixture was concentrated to dryness anddissolved in ethyl acetate (20 mL). The organic layer was washed withwater and 10% sodium bicarbonate solution and then dried over sodiumsulfate, filtered and concentrated. The residue was purified by columnchromatography using dichloromethane (96):methanol (4) as eluent to givethe hydrazone 3 (0.116 g, 92%).

A solution of the above hydrazone (3) (0.025 g, 24.7 μmol), transporter(1×, Bacar₉CCONH₂.9TFA, Bacar₇CCONH₂.7TFA, BacaCCONH₂, NH₂r₇CCONH₂.8TFA,NH₂R₇CCONH₂.8TFA) and diisopropylethylamine (1×) in anhydrousdimethylformamide (1 mL) were stirred under nitrogen at room temperaturefor 36 h when TLC indicated the complete disappearance of the startinghydrazone. Solvent was evaporated from the reaction mixture and theresidue purified by reverse phase HPLC using trifluoroacetic acidbuffered water and acetonitrile.

Yields of conjugates with various transporters:

-   -   Conjugate with Bacar₉CCONH₂.9TFA (4)—73%    -   Bacar₇CCONH₂.7TFA (5)—50%    -   BacaCCONH₂ (6)—52.9%    -   NH₂r₇CCONH₂.8TFA (7)—43.8%    -   NH₂R₇CCONH₂.8TFA (8)—62.8%

Structures of all the products were confirmed by ¹H-NMR spectra and TOFMS analysis.

Linker 2 2-(2-pyridinyldithio)ethyl hydrazine carboxylate (Scheme IIIand IV)

A solution of FK506 (1) (0.1 g, 124.4 μmol), 2-(2-pyridinyldithio)ethylhydrazine carboxylate (9) (0.091 g, 373.2 μmol) and trifluoroacetic acid(catalytic, 1 μL) in anhydrous methanol (5 mL) was stirred at roomtemperature for 16 h. The reaction was monitored by thin layerchromatography that showed almost complete disappearance of the startingmaterial. [TLC solvent system—ethyl acetate R_(f)=0.5]. The reactionmixture was concentrated to dryness and dissolved in ethyl acetate (20mL). The organic layer was washed with water and 10% sodium bicarbonatesolution and then dried over sodium sulfate, filtered and concentrated.The residue was purified by column chromatography using dichloromethane(97):methanol (3) as eluent to give the hydrazone 10 (0.091 g, 71%)

Example 18 Differential Uptake of Transporters in the GastrointestinalTract Methods

Gastrointestinal Absorption Protocol

Experiments were performed on 8- to 10-week-old female Swiss Webstermice purchased from Taconic (Germantown, N.Y.). Mice were anesthetizedwith Nembutal and a midline incision was made along the abdomen.Intestines were measured, tied off at both ends of the desired sectionwith sutures, and biotinylated peptides were injected into the lumen(approximately 100λ/inch). After a fifteen minute incubation, the tissuewas removed and the lumen was gently washed with PBS.

To determine whether CellGate transporters could enter the squamousepithelia of the oral cavity, mice were anesthetized with Nembutal,their heads tipped to one side and solutions of the biotinylatedpeptides were placed in their mouths. After fifteen minutes the liquidwas removed by pipette.

Preparation of Histological Sections of Regions of the GastrointestinalTract

Immediately following incubation with biotinylated peptides, theanesthetized rodents were sacrificed by cervical dislocation and thetied off sections of the GI tract were removed. The lumens of thevarious sections were filled with OCT using a plastic tipped syringe,immersed in OCT filled boats, and snap frozen in a 2-methyl-butane/dryice solution. Frozen sections were allowed to warm slightly and 2 mmthick sagital cuts were made using a steel razor blade. The cuts wereplaced into OCT molds and snap frozen in a 2-methyl-butane/dry icesolution. Sections (5 μm) were cut on the cryostat, fixed in acetone at4° C. for 10 minute, and allowed to air dry. After rehydration in PBSfor 5 minutes, sections were blocked with normal horse serum, washed,incubated with streptavidin-FITC (20 μg/ml) for 30 minutes, washed, andmounted with mounting medium containing propidium iodide (PI).

Tissue Culture

Caco-2 cells were acquired from ATCC, thawed, and grown in DMEMcontaining penicillin, streptomycin, glutamine, and 10% fetal bovineserum for 3-5 days to confluency (>250,000 cells/cm²) and then passagedat 60,000 cells/cm² for several passage cycles. Passage numbers 25-29were plated on Snapwell 12 mm diameter 0.4-micron pore sizepolycarbonate membranes (Corning Costar, Corning, N.Y.) at 60,000 cellsper membrane and allowed to grow for at least 21 days, with mediachanged every other day.

Cellular Uptake in Caco-2 Cells

To analyze the penetration of fluorescent oligomers of D-arginine intothe cells when in suspension, the monolayers were treated with trypsin(4.5 ml of a 0.05% solution Gibco, Grand Rapids, Mich.) and theindividual cell suspension was spun down. Cells were resuspended,counted and treated with varying concentrations of Fl aca r5, Fl aca r7,Fl aca r9 and Fl aca k9 (from 50-0.8 μM) for five minutes, washed,resuspended in 400λ PBS, 2% FBS, 40 ngPI/ml and analyzed by flowcytometry.

To analyze the ability of the fluorescent peptides to enter Caco-2 cellswhen part of a monolayer, the cells were seeded in lab-tek flaskettemicroscope slides (Nalge Nunc Int., Naperville, Ill.) in 4 ml at adensity of 60,000 cells/ml and grown for 21 days with the media beingchanged every other day. Once the monolayer was established, it wasincubated with 100 μM Fl aca r9 CONH₂ for a five minutes. The monolayerwas subsequently washed with PBS/2% FBS twice to remove labeled peptideand analyzed using fluorescent microscopy.

Transport Across Monolayers of Caco-2 Cells

The experiments analyzing whether short oligomers of D-arginine couldcross monolayers of Caco-2 cells were performed using a voltage clampamplifier and Easymount side-by-side horizontal diffusion chamber(Physiologic Instruments, San Diego, Calif.) with a 95% oxygen 5% carbondioxide gas lift system connected to a recirculating water bath.

Current measurements (I₂−I₁) were taken at five minute intervals,including 10 minutes prior to the addition of the test compounds toinsure monolayers were intact. At zero time test compounds were added atthe proper concentrations and measurements recorded. Measurements weretaken for 60 minutes at 5 minute intervals; transepithelia electricalresistance (TEER) and short circuit current (Isc) were subsequentlycalculated from these values.

Synthetic Chemistry

Cyclosporin A

The details of the CsA conjugate used in this study have been describedpreviously (Rothbard et al. Nature Medicine 6, 1253 2000). See also,FIG. 6. Briefly, CsA was conjugated to a heptamer of D-arginine througha pH sensitive linker as shown in FIG. 6A. The resultant conjugate isstable at acidic pH but at pH>7 it undergoes an intramolecularcyclization involving addition of the free amine to the carbonyladjacent to CsA (FIG. 6B), which results in the release of unmodifiedCsA.

Taxol

Taxol was treated with α-chloro acetic anhydride delivering the C-2′chloro acetyl derivative 12 in essentially quantitative yield.

The halogen was displaced by the thiol of the N-terminal (L) cysteinecontaining heptamer of D-arginine. Conjugations were performed at roomtemperature in DMF in the presence of DIEA. The final products wereisolated by RP-HPLC and lyophilized to yield TFA salts, which were veryhygroscopic and readily dissolve in water.

Compound 13 was designed to release taxol via a nucleophilic attack ofthe N-terminal nitrogen onto the C2′ ester carbonyl. The protonationstate of this nitrogen is crucial for this mechanism, since only thefree amine will be capable of this release. Additionally, bothconjugates share a common α-hetero atom substituted acetate moietymaking them susceptible to simple ester hydrolysis. This offers anadditional release pathway.

Intracolonic Injections

Wistar rats, females of approximately 200-300 g (Simonsen, Gilroy,Calif.), were anesthetized, their abdomens were shaved, midlineincisions were made, and a one-half inch section of the ascending colonin each animal was tied off with sutures. Taxol and cyclosporin (5mg/kg) were injected as solutions in a 1:1 v/v Cremophor EL:ethanolmixture, whereas equivalent molar amounts of the r7 conjugates wereinjected in PBS. In all cases, the volume injected was approximately500λ. The colon was placed back into the cavity, and the incision wasclosed using sutures.

Blood samples were taken from the tail vein at time zero (prior to druginjection), and every thirty minutes for the duration of the experiment,which was empirically determined, with the exception of one animal thatexpired after ninety minutes. Clotting was inhibited by transferring theblood to glass tubes containing 100 k of 0.5% EDTA, and the blood wasfrozen.

Drug Extraction from Whole Blood and HPLC MS MS Analysis

Either taxol or cyclosporin was extracted from the whole blood using amodification of literature procedures. Briefly, whole blood (100λ) wastransferred to a screw capped glass tube containing five mls of diethylether. The sample was vortexed vigorously for two minutes, centrifuged,and frozen in dry ice/methanol. The ethereal layer was transferred toanother glass tube and the ether was evaporated. In the case ofcyclosporin, the residue was resuspended in 1.5 mls of methanol, water,acetonitrile (3:2:1), while for taxol the residue was resuspended in 1.5mls methanol:acetonitrile (1:1). Samples were placed into a Perkin-Elmerseries 200 autosampler and sequentially injected onto a C18 reversecolumn at 70° C. connected to a Shimadzu HPLC system, eluted with 70%methanol, 30% aqueous ammonium formate buffer, and the effluent wasanalyzed on a PE Sciex API 3000 tandem mass spectrometer. Known amountsof either cyclosporin A (10-1000 ng/ml) or taxol (1-1000 ng/ml) wereadded to whole blood and extracted as previously described to generatestandard curves. Cyclosporin A was monitored by the two transitions fromboth 1220 to 1203 daltons and 1220 to 100 daltons. The 1220 speciescorresponds to the cyclosporin A+ammonia, the 1203 is protonatedcyclosporin, while 100 is a known fragment. Taxol was monitored by thetwo transitions from both 872 to 855 daltons and 872 to 110 daltons. The872 species corresponds to the ammonium adduct of taxol, the 855 is theprotonated parent compound, whereas 110 is the predominant fragment seenin the second quadrupole.

The total amount of cyclosporin A and taxol in the samples wasdetermined by comparing the integrated area of the appropriate peak withvalues established by the standard curves.

Results

Differential Uptake of Transporters in the Gastrointestinal Tract

Short oligomers of D-arginine have been shown to cross rapidly andefficiently the plasma membrane of a large variety of cell lines grownin suspension. In addition, they have been shown to penetrate multiplelayers of the skin when applied topically, multiple layers ofendothelial and smooth muscle cells of veins and arteries when injectedintravenously, and multiple cell layers of lung tissue when inhaled. Todetermine whether these compounds could enter the nonkeratinizedepithelia of the gastrointestinal tract, sections of the small and largeintestines of fasted mice were tied off and solutions of biotinylatednonamers of D-arginine, bio aca r9 CONH₂, (100 μM) were injected. Afterfifteen minutes of exposure, the relevant section of tissue wasdissected, frozen, sectioned, and stained with Streptavidin fluoresceinto define the location of the biotinylated peptide, and propidium iodideto counterstain all the nuclei in the section.

When injected into the lumen of murine duodenum no detectable stainingover background was observed. Poor staining also was seen when thebiotinylated peptide was injected into the jejunum. If multiple sectionswere scanned detectable staining was seen on the tips of some villi. Thefirst sign of uniform staining was observed when sections of the ileumwere analyzed. The staining was localized to the tips of the microvilliand did not extend into the crypt cells. Although seen throughout allsections of the ileum, the observed fluorescence did not approach thelevel of intensity previously observed in the skin, lungs, or theendothelial cells of arteries or veins.

The relatively poor staining of the small intestine markedly differedfrom that seen when regions of the colon were examined. In both theascending and transverse sections of the large intestine, biotinylatednonamers of arginine stained all surface areas of the villi, andpenetrated several cell layers, reminiscent of the intense staining ofthe epidermis and dermis when applied topically. In addition, the cryptcells were heavily stained in the colonic sections and evidence forpenetration of the full thickness of the section was observed in severalareas.

The histological analysis demonstrated that the uptake of bio r9 intothe nonkeratinized epithelia layers of the GI tract variedsignificantly, with uptake increasing with distance from the gastricpylorus. The staining observed in the ileum was greater than that seenin the jejunum, which was greater than the duodenum. The greateststaining was seen in the ascending and transverse regions of the colon.Without intending to limit the invention to a particular theory ormechanism, one theory is that the composition and amount of mucus liningthe epithelia might be an important factor in the differential staining.

Cellular Uptake into Caco-2 Cells

When Caco-2 cells, commercially-available human colon cells, wereincubated in suspension with varying amounts of fluorescently labeledpentamers, heptamers, and nonamers of D-arginine (Fl aca r5, Fl aca r7,Fl aca r9) or nonamers of lysine (Fl aca k7), and analyzed by flowcytometry, a pattern similar to that previously seen in a variety ofother suspension cells (Mitchell et al. Peptide Research 56, 318 (2000))was observed (FIG. 32). Uptake of the fluorescent peptides increasedwith arginine content with r9 being more effective than r7, which wasmore effective than r5. All the polymers of arginine entered cells moreeffectively than the nonamers of lysine. Both the rate, the relativeamount of fluorescence, and the lack of apparent efflux of theinternalized fluorescent peptides over an extended period of time(several hours) were reminiscent of earlier experiments withlymphocytes.

To confirm that the rapid penetration of the short oligomers of arginineinto Caco-2 cells was not an artifact seen only when the cells were insuspension, Fl aca r9 CONH₂ (50 μM) was incubated for five minutes withCaco-2 cells grown as a monolayer on a microscope slide, washed, andanalyzed by fluorescent microscopy. Consistent with the flow cytometryanalysis of the suspension cells, virtually every cell in the monolayerwas fluorescent after five minutes.

The ability of the peptides to rapidly enter Caco-2 cells was firmlyestablished by placing a monolayer of Caco-2 cells as a membrane in adiffusion chamber and exposing it to fluorescent transporter drugconjugates between taxol and oligomers of arginine of different length.Varying concentrations of the taxol conjugates (50-0.08 μM) were addedto the apical side and exposed to the monolayer of Caco-2 cells forthree minutes. The membrane was removed from the apparatus, the cellstrypsinized, and the resulting cell suspension was analyzed by flowcytometry (FIG. 33). As with the peptides alone, the drug conjugatesquickly and efficiently entered the cells composing the monolayer withthose containing more arginine subunits being more effective.

Without intending to limit the theory of the invention, theseexperiments provide strong support for the hypothesis that shortoligomers of arginine, either conjugated to fluorescein or therapeuticdrugs, such as taxol, rapidly enter Caco-2 cells both in suspension andwhen grown in monolayers.

Transport Across Caco-2 Monolayers

Crossing the luminal membrane of the gut epithelia is necessary toincrease blood levels of a delivered (e.g., buccal administered) drug.To determine whether the transporters could cross the gut epithelia,monolayers of Caco-2 cells were grown in culture and placed as amembrane in a commercially available diffusion chamber. The integrity ofthe membranes was established by demonstrating that the transepithelialelectronic resistance (TEER) was always greater than 100 ohm cm² (FIG.34). Such a pattern of stable resistance only is observed when themembrane is intact with no significant spaces between the cells.

Additional evidence that the membrane was both viable and contiguous wasthat Lucifer Yellow (200 μM) was not transported across the monolayer,whereas hydrocortisone was transported at amounts consistent withpublished reports (FIG. 35).

When a variety of fluorescent oligomers of D- or L-arginine, rangingfrom four to 15 subunits, were placed in the apical chamber in multipleexperiments with a large number of membranes, none were significantlytransported into the basolateral chamber (FIG. 35).

Taken together, the data presented herein are consistent with the modelthat short oligomers of arginine either conjugated to deliveredcompounds such as fluorescein or taxol rapidly enter but are nottransported across Caco-2 cell monolayers. This model also implies thatthe transporters of the invention do not enter Caco-2 cells byendosomes, which are transported across colonic epithelium and excretedon the basolateral side by a well understood pathway. CellGatetransporters appear to enter the cytosol directly and do not have a highrate of efflux on the basolateral side.

Measurement of Drug Blood Levels after Intracolonic Injections

Although short oligomers of arginine labeled with fluorescein, eitheralone, or when conjugated to either taxol or cyclosporin, were unable tocross a monolayer of Caco-2 cells in vitro, the cell line may notprecisely mimic the in vivo behavior of the colon. Furthermore, thepattern of fluorescence seen in colon tissue after incubation with shortoligomers of arginine demonstrated that the peptides penetrated intolayers known to be vascularized.

To determine whether the transporters of the invention could enhance thedelivery of orally administered drugs, releasable conjugates ofcyclosporin A and taxol were injected intracolonically and the resultingblood levels of the released drugs were measured by LC MS MS.

The first experiment was designed to measure blood levels of CsA afterintracolonic injection of 5 mg/kg of CsA in Cremophor EL:ethanolcompared with an equimolar amount of a releasable r7 conjugate of CsAdissolved in phosphate buffered saline. In the case of the parent drug,blood levels rose to approximately 25 ng/ml of CsA after thirty minutes,and then rapidly fell off to levels close to baseline levels (FIG. 36).No further data was obtained because after 90 minutes the animal died.In contrast, when an equimolar amount of CsA-r7 conjugate was injected,detectable levels of CsA in the blood were observed only after 3 hours(FIG. 36). To determine whether the altered pharmacokinetics of CsA whenconjugated to a short oligomer of arginine was reproducible, a third ratwas injected with 10 mg/kg equivalent of the water soluble, releasableconjugate. The rate of uptake of CsA in the blood of this animalresembled the animal injected with 5 mg/kg of the conjugate. With moreconjugate administered a small increase was seen at 30 minutes, butlarger amounts appeared in the blood only after two hours, with bloodlevels approaching 45 ng/ml after three hours. The overall pattern wassimilar in the two animals injected with the conjugate. In both casesthe overall amount of CsA measured in the circulation was significantlygreater than observed when CsA was injected.

The half-life of the CsA conjugate was approximately ninety minutes,which was consistent with the delay in the appearance of CsA in theblood relative to the parent compound. This fact combined with thehistological data demonstrating rapid and efficient uptake in thecolumnar epithelium of the colon and the failure of nonreleasableconjugates of CsA or taxol to cross monolayers of colonic cells invitro, leads to a sensible and simple model describing the phenomena.Without intending to limit the invention to a particular mechanism ortheory, it appears that the conjugates enter the columnar epithelia ofthe colon with greater efficiency and more rapidly than CsA injected inCremophor, but were retained in the epithelial cells and did not crossthe endothelial cells surrounding the capillaries until the conjugatehydrolyzed. Once released, the CsA freely diffused, or was activelytransported, into the blood.

To test this hypothesis, taxol and two different r7-taxol conjugateswere injected into the colon and blood levels of the drug were measuredat thirty minute intervals. The two r7-taxol conjugates hadsignificantly different half-lives (10 minutes and 5 hours) and wereused to test the hypothesis that the hydrolysis of the drug conjugatewas the rate limiting step in appearance of the drug in the circulation.If true, the premise predicted that the r7-taxol conjugate with the tenminute half life would release taxol in the epithelia so that it couldbe detected in the circulation at the thirty minute time point. Incontrast, taxol should not be released from the more stable r7 conjugate(t_(1/2)=5 hours) and should not be detected in the blood samples takenduring the experiment.

This premise was supported by the appearance of taxol in the blood (FIG.37). Detectable levels of taxol did not appear in the blood wheninjected in the colon until 2.5 hours with subsequent waves at 4 and 5.5hours. The oscillating levels of taxol in the blood as a function oftime were consistent with published studies with the pattern beingrationalized to the ability of Cremophor to sequester some of thematerial and act as a timed release vehicle. In contrast, when a labile,water soluble r7 conjugate of taxol (13) was administered, greater than200 ng/ml of taxol was observed at the earliest time point (30 minutes)which continued to increase up to 1 hour, at which point the levelsslowly diminished out to five hours. As predicted, injection of the morestable, water soluble r7 conjugate of taxol (14) did not result insignificant blood levels of taxol within five hours. These data supportthe hypothesis that transporters of the invention enter, but do nottransverse the colonic epithelium. They can improve oral bioavailabilityof drugs both by dramatically improving water solubility and byincreasing the rate of uptake of the conjugate in the colon. Ultimatedelivery of the drug into the blood stream appears to depend on thedrug's inherent ability to diffuse through biological membranes and therate of release from the conjugate.

Discussion

A significant discovery in the experiments described herein is thefailure of the peptides to enter the epithelia of the duodenum. Thisrepresents the first example of the transporters not entering a celltype or tissue. The pattern of increasing uptake the further the tissueextended from the gastric pylorus was intriguing, most likely due to agradient of an inhibitor, such as a negatively charged mucus. Thefailure to stain the upper regions of the small intestine was in starkcontrast with the intense staining in the colon, leading to thespeculation that transporters of the present invention could be used forselective delivery of therapeutics to the colon. Not only were allluminal surfaces of the colon highly fluorescent, but the stainingpattern also revealed that the biotinylated compounds were able topenetrate multiple cell layers and reach vascularized regions of thetissue, suggesting that the transporters should enhance transport intothe bloodstream.

However, separate studies using monolayers of the colonic cell line,Caco-2, suggests an alternative mechanism. In the Caco-2 system thetransporters rapidly entered, but did not traverse the monolayer. Thereare several examples of the transporters exhibiting high rates oftransport into, but limited rates of efflux from cells and tissue. Thisis the case for all suspension cell lines examined to date, the beststudied being the human T cell line, Jurkat. Short oligomers ofD-arginine rapidly enter these cells and exhibit very low exit rates,losing less than 5% of the fluorescent signal after one hour ofincubation at 37° C. An even more relevant example of this phenomenon isthe low levels of CsA measured in the blood stream of mice receivingmultiple topical treatments of a releasable CsA r7 conjugate. As in thecase for the colon, staining patterns in sections of skin treated with abiotinylated analog of the CsA conjugate established that the drugpenetrated into highly vascularized regions of the dermis. In addition,staining with monoclonal antibodies established that one of theprominent cell types in the dermis that were highly fluorescent were theendothelial cells of the capillaries. Nevertheless, detectable levels ofCsA in the blood of these animals were never observed even after tendays of treatment with a 4% ointment applied twice a day. Thisobservation, although anecdotal, is consistent with results betterstudied in this report.

The ability of the peptides to enter and subsequently exit multiplelayers of cells in the skin, the lungs, and the cardiovascular system isin marked contrast to their inability to exit from lymphocytes or Caco-2cells. Without intending to limit the present invention to a particulartheory or mechanism, one difference is the cells through which thepeptides rapidly penetrate are connected by tight junctions and othermembrane structures inherent in tissue architecture, whereas theindividual cells in suspension and perhaps the side of the membrane ofendothelial cells contacting the bloodstream lack these features. Ifstructures such as tight or gap junctions modify the surrounding lipidto permit exit of the transporters, then they should be able to diffuserapidly throughout a tissue, such as skin or the colon, but not betransported into the bloodstream. Consistent with this hypothesis is themodel constructed to explain the variations in the rates of appearanceof taxol and CsA in the circulation in this report. In this model, theshort oligomers of arginine greatly promoted uptake into columnarepithelium of the colon or the squamous epithelium of the cheek, but didnot transport the drug into the bloodstream. The blood levels observedwere the result of the diffusion, or active transport, of the drug outof the epithelium into the circulation after hydrolysis of theconjugate.

Example 18 Buccal Delivery of Transporter Conjugates

Buccal delivery of taxol and CsA involved adding a concentrated solution(250% of 5 mg/kg) to the oral cavity of an anesthetized rat lying on itsside. Blood samples were taken from the tail vein at time zero (prior todrug injection), and every tinny minutes for the duration of theexperiment, which was empirically determined. Clotting was inhibited bytransferring the blood to glass tubes containing 100λ of 0.5% EDTA, andthe blood was frozen.

The capacity of the transporters to enter the squamous epithelial layersof the oral cavity was examined. A mouse was anesthetized, its head wastipped so that a solution of biotinylated r9 could be administered. Theanimal was kept in this position for fifteen minutes, at which time itwas sacrificed, and the tongue and cheek were dissected, frozen,sectioned, stained with streptavidin-fluorescein, and counterstainedwith propidium iodide. In both the cheek and the tongue the biotinylatedpeptides quickly and efficiently penetrated multiple layers of theepithelia and penetrated deep into the interior layers of the tissue,reminiscent of the staining of both the epidermis and dermis of theskin.

In a second experiment, rats were anesthetized and solutions of bothtaxol and CsA (5 mg/kg) in Cremophor EL:ethanol 1:1 or equimolar amountsof the corresponding r7 conjugates of these drugs in PBS were simplyincubated in the cheek pouch of the animal for the duration of theexperiment. LC MS was used to determine the blood levels of only taxoland the fast releasing r7 conjugate. Both the amount and the kinetics ofappearance of taxol in the blood when administered in the oral cavity(FIG. 38) differed from when it was injected into the colon. Buccaladministration of taxol conjugates appeared to be less effective, withless than one eighth of the amount of taxol being observed in thecirculation compared with intracolonic injection. Another difference wasthe rate of appearance of the unmodified drug. When administered in theoral cavity, taxol appeared in the blood stream by the first time point,whereas in the colonic injection detectable amounts of taxol did notappear for several hours. Even though there was no difference betweentaxol and the r7-taxol conjugate in the appearance of taxol in thecirculation in buccal administration, approximately twice as muchmaterial reached the circulation when the conjugated was used.

Example 19 Ocular Delivery of Transporter Conjugates

The ability of the transporters of the invention to penetrate thetissues of the eye was examined. Biotinylated r8 was both injected intothe eyes of rabbits and also applied as eyedrops to the outside of theeye.

Briefly, 5 drops of a 1 mM solution of biotinylated r8 in PBS wasapplied to both eyes of a rabbit and allowed to incubate 15 minutes. Theanimal was sacrificed and one eye was dissected intact with adjacenttissue, whereas the other was separated into each of its componentparts, frozen and separately sectioned, stained withstreptavidin-fluorescein and counterstained with propidium iodide.results demonstrated staining in the cornea and eyelid, but not thelens.

50 microliters of a 10 mM solution of biotinylated r8 in PBS wasinjected into the vitreous humor of another animal and the animal wassacrificed 30 minutes later and the injected eye was dissected. Againone orbital was frozen intact while the other was dissected and thecomponents separately frozen. Results from the injection experimentsdemonstrated that all interior surfaces of the orb were stained.

Example 20

This example illustrates the conjugation of cyclosporin to a transportmoiety using a pH sensitive linking group (see FIGS. 6A and 9B).

In this example, cyclosporin is converted to its α-chloroacetate esterusing chloroacetic anhydride to provide 6ii (see FIG. 6). The ester 6iis then treated with benzylamine to provide 6ii. Reaction of the aminewith Boc-protected iminodiacetic acid anhydride provides the acid 6iiiwhich is then converted to an activated ester (6iv) with N-hydroxysuccinimide. Coupling of 6iv with L-Arginine heptamer provides theBOC-protected conjugate 6v, which can be converted to conjugate 6vi byremoval of the BOC protecting group according to established methods.

Transport moieties having arginine groups separated by, for example,glycine, ε-aminocaproic acid, or γ-aminobutyric acid can be used inplace of the arginine heptamer in this and in the following examplesthat show oligoarginine transport groups.

Example 21

This example illustrates the conjugation of acyclovir to a transportmoiety.

a. Conjugation of Acyclovir to r₇CONH₂

This example illustrates the conjugation of acyclovir to r₇CONH₂ via thelinking group:

i) Preparation of Acyclovir α-Chloroester:

A solution of acyclovir (100 mg, 0.44 mmol), dimethylaminopyridine (5.4mg, 0.044 mmol) and chloroacetic anhydride (226 mg, 1.32 mmol) indimethylformamide (9 mL) was stirred at room temperature for 18 h. Thedimethylformamide was removed by evaporation. The crude product waspurified by reverse-phase HPLC (22 mm×250 mm C-18 column, a 5-25%CH₃CN/H₂O gradient with 0.1% trifluoroacetic acid, 214 and 254 nm UVdetection) and lyophilized. The product was obtained as a white powder(62 mg, 47%). ¹H NMR (300 MHz, DMSO-d₆) δ 10.67 (s, 1H), 7.88 (s, 1H),6.53 (s, 1H), 5.27 (s, 2H), 4.35 (s, 2H), 4.21 (t, J=3 Hz, 2H), 3.70 (t,J=3 Hz, 2H); ¹³C NMR (75 MHz, DMSO-d₆) δ 168.1, 157.6, 154.8, 152.3,138.6, 117.1, 72.7, 67.1, 65.2, 41.8; TOF-MS (m/z): 302.0 [M+H].

ii) Conjugation of Acyclovir α-Chloro Ester to H₂N—C-r7-CONH₂

A solution of acyclovir α-chloroester (7 mg, 0.024 mmol), H₂N—C-r7-CONH₂(50 mg, 0.024 mmol) and diisopropylethylamine (6.4 μL, 0.036 mmol) indimethylformamide (1 mL) was stirred for 18 h. The dimethylformamide wasremoved by evaporation. The crude product was purified by reverse-phaseHPLC (22 mm×250 mm C-18 column, a 5-25% CH₃CN/H₂O gradient with 0.1%trifluoroacetic acid, 214 and 254 nm UV detection) and lyophilized. Thedesired product was obtained as a white powder (24 mg, 69%). TOF-MS(m/z): 494.6 [(M+H)/3], 371.0 [(M+H)/4].

The yield could be increased by using 10 molar equivalents ofdiisopropylethylamine rather than 1.5 molar equivalents. Product wasagain obtained as a white powder (79%). TOF-MS (m/z): 508.7 [(M+H)/3],381.5 [(M+H)/4], 305.5 [(M+H)/5].

b. Conjugation of Acyclovir to a Biotin-Containing Derivative ofr₅-Cys-CONH₂

Reactions were carried out as illustrated above, using the synthetictechniques provided in the examples above.

i) Biotin-aminocaproic acid-r5-Cys(acyclovir)-CONH₂ was obtained as awhite powder (36%). TOF-MS (m/z): 868.2 {(M+2 TFA)/2], 811.2 [(M+1TFA)/2], 754.1 [(M+1 TFA)/3], 503.0 [(M+H)/3], 377.4 [(M+H)/4].

Similarly,

ii) Biotin-aminocaproic acid-r7-C(acyclovir)-CONH₂— was obtained as awhite powder (33%). TOF-MS (m/z): 722.1 [(M+3 TFA)/3], 684.6 [(M+2TFA)/3], 607.1 [(M+H)/3], 455.5 [(M+H)/4], 364.8 [(M+H)/5], 304.3[(M+H)/6].

Example 22

This example illustrates the conjugation of hydrocortisone to atransport moiety.

a. Conjugation of Hydrocortisone to r₇CONH₂

i) Preparation of Hydrocortisone α-Chloroester:

To a solution of hydrocortisone (500 mg, 1.38 mmol), scandium triflate(408 mg, 0.83 mmol) and chloroacetic anhydride (708 mg, 4.14 mmol) indry THF was added dimethylaminopyridine (506 mg, 4.14 mmol). Thesolution turned bright yellow upon addition of dimethylaminopyridine.After 30 min the solvent was evaporated off and the crude material takenup into ethyl acetate (100 mL). The ethyl acetate layer was washed with1.0 N HCl and brine. The organic phase was collected, dried (Na₂SO₄) andevaporated to provide the product as a white solid (533 mg, 88%). ¹H NMR(300 MHz, DMSO-d₆) δ 5.56 (s, 1H), 5.46 (s, 1H), 5.20 (d, J=18 Hz, 1H),4.85 (d, J=18 Hz, 1H), 4.51 (s, 2H), 4.37 (br s, 1H), 4.27 (br s, 1H),2.54-2.33 (m, 2H), 2.22-2.03 (m, 3H), 1.99-1.61 (m, 8H), 1.52-1.24 (m,5H), 1.02-0.98 (d, J=12 Hz, 1H), 0.88-0.85 (d, J=9 Hz, 1H), 0.77 (s,3H); ¹³C NMR (75 MHz, DMSO-d₆) δ 205.4, 198.8, 173.0, 167.6, 122.3,89.5, 69.7, 67.3, 56.4, 52.4, 47.8, 41.6, 39.7, 35.0, 34.3, 34.0, 33.6,32.3, 32.0, 24.2, 21.3, 17.4; TOF-MS (m/z): 439.1 (M+H).

-   (Reference for acetylation—Zhao, H.; Pendri, A.; Greenwald, R. B. J.    Org. Chem. 1998, 63, 7559-7562.)    ii) Coupling to R′NH-Cys-r₇-CONH₂

A solution of hydrocortisone α-chloroester (31 mg, 0.071 mmol),H₂N—C-r7-CONH₂ (150 mg, 0.071 mmol) and diisopropylethylamine (15 μL,0.085 mmol) in dimethylformamide (1 mL) was stirred for 18 h. Thedimethylformamide was evaporated off. The crude product purified byreverse-phase HPLC (22 mm×250 mm C-18 column, a 5-30% CH₃CN/H₂O gradientwith 0.1% trifluoroacetic acid, 214 and 254 nm UV detection) andlyophilized. The desired product was obtained as a white powder (25 mg,14%). TOF-MS (m/z): 1037.4 [(M+4 TFA)/2], 616.1 [(M+2 TFA)/3], 578.3[(M+1 TFA)/3], 540.5 [(M+H)/3], 405.7 [(M+H)/4], 324.5 [(M+H)/5].

The use of 10 molar equivalents of diisopropylethylamine rather than 1.2molar equivalents provided the desired product as a yellow powder (52%yield). TOF-MS (m/z): 887.0 [(M+1TFA)/2], 830.6 [(M+H)/2], 553.7[(M+H)/3], 415.5 [(M+1-1)/4].

b. Conjugation of Hydrocortisone to a Biotin-Containing Derivative ofr₅-Cys-CONH₂

Reactions were out as illustrated above, using the synthetic techniquesprovided in the examples above.

i) Biotin-aminocaproic acid-r5-C(hydrocortisone)-CONH₂— Used 10 molarequivalents of diisopropylethylamine rather than 1.2 molar equivalents.Product a white powder (65%). TOF-MS (m/z): 880.7 [(M+1 TFA)/2], 548.7[(M+H)/3].

ii) Biotin-aminocaproic acid-r7-C(hydrocortisone)-CONH₂— Used 10 molarequivalents of diisopropylethylamine rather than 1.2 molar equivalents.Product a white powder (36%). TOF-MS (m/z): 692.3 [(M+1 TFA)/3], 652.8[(M+H)/3], 520.0 [(M+1 TFA)/4], 490.0 [(M+H)/4], 392.5 [(M+H)/5].

Example 23

This example illustrates the conjugation of taxol to a transport moiety.

a. Conjugation of Taxol to r₂-CONH₂

This example illustrates the application of methodology outlined aboveto the preparation of a taxol conjugate (see FIG. 12).

i) Preparation of a Taxol α-Chloroacetate Ester

Taxol was treated with α-chloro acetic anhydride providing the C-2′chloro acetyl derivative 12i in essentially quantitative yield.

ii) Formation of Taxol Conjugate

The halogen atom of the chloroacetate ester was displaced by the thiolof an N-terminal (L) cysteine containing heptamer of arginine. To avoiddegradation of the transporter entity by proteases in-vivo, D-argininewas used as the building unit. Conjugation reactions were performed atroom temperature in DMF in the presence of diisopropylethylamine. Thefinal products were isolated by RP-HPLC and lyophilized to whitepowders. It is important to note that the native conjugate (R═H) isisolated as its TFA salt at the cysteine primary amine. The conjugatesare generally quite hygroscopic and readily dissolve in water.

The conjugate wherein R═H was designed to release the parent drug via anucleophilic attack of the N-terminal nitrogen onto the C2′ estercarbonyl. The protonation state of this nitrogen is crucial for thismechanism, since only the free amine will be capable of this release.Additionally, both conjugates share a common α-hetero atom substitutedacetate moiety making them susceptible to simple ester hydrolysis. Thisoffers an additional release pathway.

Example 24

This example illustrates two methods of linking active agents totransport moieties. Illustration is provided for retinoic acidderivatives linked to poly-D-Arg derivatives but can be applied tolinkages between other biological agents and the transport moieties ofthe present invention.

a. Linkage Between a Biological Agent Having an Aldehyde FunctionalGroup

This example illustrates the preparation of a conjugate between anonamer of D-arginine (H₂N-r₉-CO₂H.10TFA) and either all trans-retinalor 13-cis-retinal. FIG. 40 provides a schematic presentation of thereactions. As seen in FIG. 40, condensation of either retinal withH₂N-r₉-CO₂H.10TFA in MeOH in the presence of 4 Å molecular seives atroom temperature for four hours results in the formation of a Schiffbase-type linkage between the retinal aldehyde and the amino terminalgroup. Purification of the conjugate can be accomplished by filteringthe molecular sieves and removing methanol under reduced pressure.

b. Conjugation of Retinoic Acid to r₇-CONH₂

This example illustrates the preparation of a conjugate between retinoicacid and r₇-CONH₂ using the linking group

Here, preparation of the conjugate follows the scheme outlined in FIG.41. In this scheme, retinoic acid (41ii) is first combined with thechloroacetate ester of 4-hydroxymethyl-2,6-dimethylphenol (41i) toprovide the conjugate shown as 41iii. Combination of 41i with retinoicacid in methylene chloride in the presence of dicyclohexylcarbodiimideand a catalytic amount of 4-dimethylaminopyridine provided the retinoidderivative 41iii in 52-57% yield. Condensation of 41iii withH₂NCys-r₇CONH₂.8TFA in the presence of diisopropylethylamine (DMF, roomtemperature, 2 h) provides the desired conjugated product 41iv.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

What is claimed is:
 1. A method of enhancing delivery of a compoundacross the blood-brain barrier of an animal, the method comprisingcontacting the blood-brain barrier with a conjugate consisting of thecompound and a delivery-enhancing transporter, at a concentrationeffective for delivering a therapeutically effective amount across theblood-brain barrier, wherein the delivery-enhancing transporter consistsof 5 to 50 amino acids that comprises 5 to 25 arginine residues oranalogs thereof, thereby increasing delivery of the conjugate across theblood-brain barrier compared to delivery of the compound in the absenceof the delivery-enhancing transporter, and wherein the conjugate has astructure selected from the group consisting of structures 3, 4, and 5,as follows:

wherein: R¹ comprises the compound; X is a linkage formed between afunctional group on the compound and a terminal functional group on thelinking moiety; Y is a linkage formed from a functional group on thetransport moiety and a functional group on the linking moiety; A is N orCH; R² is hydrogen, alkyl, aryl, acyl, or allyl; R³ comprises thedelivery-enhancing transporter; R⁴ is S, O, NR⁶ or CR⁷R⁸; R⁵ is H, OH,SH or NHR⁶; R⁶ is hydrogen, alkyl, aryl, acyl or allyl; R⁷ and R⁸ areindependently selected from hydrogen or alkyl; k and m are eachindependently selected from 1 and 2; and n is 1 to
 10. 2. The method ofclaim 1, wherein delivery of the conjugate across the blood-brainbarrier is increased at least two-fold compared to delivery of thecompound in the absence of the delivery-enhancing transporter.
 3. Themethod of claim 1, wherein delivery of the conjugate across theblood-brain barrier is increased at least ten-fold compared to deliveryof the compound in the absence of the delivery-enhancing transporter. 4.The method of claim 1, wherein X is selected from the group consistingof —C(O)O—, —C(O)NH—, —OC(O)NH—, —S—S—, —C(S)O—, —C(S)NH—, —NHC(O)NH—,—SO₂NH—, —SONH—, phosphate, phosphonate, phosphinate, and CR⁷, R⁸,wherein R⁷ and R⁸ are each independently selected from the groupconsisting of H and alkyl.
 5. The method of claim 1, wherein theconjugate comprises structure 3, Y is NH, and R² is methyl, ethyl,propyl, butyl, allyl, benzyl or phenyl.
 6. The method of claim 1,wherein R² is benzyl; k, m, and n are each 1, and X is —OC(O)—.
 7. Themethod of claim 1, wherein the conjugate comprises structure 4; R⁴ is S;R⁵ is NHR⁶; and R⁶ is hydrogen, methyl, allyl, butyl or phenyl.
 8. Themethod of claim 1, wherein the conjugate comprises structure 4; R⁵ isNHR⁶; R⁶ is hydrogen, methyl, allyl, butyl or phenyl; and k and m areeach
 1. 9. A method of enhancing delivery of a compound across theblood-brain barrier of an animal, the method comprising contacting theblood-brain barrier with a conjugate comprising the compound and adelivery-enhancing transporter, at a concentration effective fordelivering a therapeutically effective amount across the blood-brainbarrier, wherein the delivery-enhancing transporter consists of 5 to 50amino acids that comprises 5 to 25 arginine residues or analogs thereof,thereby increasing delivery of the conjugate across the blood-brainbarrier compared to delivery of the compound in the absence of thedelivery-enhancing transporter, and wherein the conjugate comprisesstructure 6 as follows:

wherein: R¹ comprises the compound; X is a linkage formed between afunctional group on the compound and a terminal functional group on thelinking moiety; Y is a linkage formed from a functional group on thetransport moiety and a functional group on the linking moiety; Ar is anaryl group having the attached radicals arranged in an ortho or paraconfiguration, which aryl group can be substituted or unsubstituted; R³comprises the delivery-enhancing transporter; R⁴ is S, O, NR⁶ or CR⁷,R⁸; R⁵ is H, OH, SH or NHR⁶; R⁶ is hydrogen, alkyl, aryl, arylalkyl,acyl or allyl; R⁷ and R⁸ are independently selected from hydrogen oralkyl; and k and m are each independently selected from 1 and
 2. 10. Themethod of claim 9, wherein X is selected from the group consisting of—C(O)O—, —C(O)NH—, —OC(O)NH—, —S—S—, —C(S)O—, —C(S)NH—, —NHC(O)NH—,—S0₂NH—, —SONH—, phosphate, phosphonate phosphinate, and CR⁷, R⁸,wherein R⁷ and R⁸ are each independently selected from the groupconsisting of H and alkyl.
 11. The method of claim 9, wherein R⁴ is S;R⁵ is NHR⁶; and R⁶ is hydrogen, methyl, allyl, butyl or phenyl.
 12. Amethod of enhancing delivery of a compound across the blood-brainbarrier of an animal, the method comprising contacting the blood-brainbarrier with a conjugate consisting of the compound and at least twodelivery-enhancing transporters, at a concentration effective fordelivering a therapeutically effective amount across the blood-brainbarrier, wherein each of the at least two delivery-enhancing transporterconsists of 5 to 50 amino acids that comprises 5 to 25 arginine residuesor analogs thereof, thereby increasing delivery of the conjugate acrossthe blood-brain barrier compared to delivery of the compound in theabsence of the delivery-enhancing transporter, and wherein the conjugatehas a structure selected from the group consisting of structures 3, 4,and 5, as follows:

wherein: R¹ comprises the compound; X is a linkage formed between afunctional group on the compound and a terminal functional group on thelinking moiety; Y is a linkage formed from a functional group on thetransport moiety and a functional group on the linking moiety; A is N orCH; R² is hydrogen, alkyl, aryl, acyl, or allyl; R³ comprises thedelivery-enhancing transporter; R⁴ is S, O, NR⁶ or CR⁷R⁸; R⁵ is H, OH,SH or NHR⁶; R⁶ is hydrogen, alkyl, aryl, acyl or allyl; R⁷ and R⁸ areindependently selected from hydrogen or alkyl; k and m are eachindependently selected from 1 and 2; and n is 1 to
 10. 13. The method ofclaim 1, wherein at least one arginine is a D-arginine.
 14. The methodof claim 1, wherein all of the arginines are D-arginines.
 15. The methodof claim 1, wherein at least 70 percent of the amino acids that comprisethe delivery-enhancing transporter are arginines or arginine analogs.16. The method of claim 1, wherein the delivery-enhancing transporter isseven contiguous D-arginines.
 17. The method of claim 1, wherein thecompound is a therapeutic agent.
 18. The method of claim 1, wherein thecompound is a diagnostic agent.
 19. The method of claim 1, wherein thecompound is an antibody or an antibody fragment.
 20. The method of claim1, wherein the compound is a therapeutic protein.
 21. The method ofclaim 1, wherein the conjugate is administered intravenously, topically,subcutaneously, transcutaneously, intramuscularly, orally, buccally, atan intra-joint site, parenterally, peritoneally, intranasally, byinhalation, or via administration to or at the skin, lungs,gastrointestinal tract, anal region, vagina, eye, or ear.
 22. The methodof claim 21, wherein the conjugate is administered intravenously. 23.The method of claim 21, wherein the conjugate is administeredsubcutaneously.
 24. The method of claim 21, wherein the conjugate isadministered topically.
 25. The method of claim 24, wherein theconjugate is administered transdermally.